Binding domain-immunogloubulin fusion proteins

ABSTRACT

The invention relates to novel binding domain-immunoglobulin fusion proteins that feature a binding domain for a cognate structure such as an antigen, a counterreceptor or the like, a hinge region polypeptide having either zero or one cysteine residue, and immunoglobulin CH2 and CH3 domains, and that are capable of ADCC and/or CDC while occurring predominantly as monomeric polypeptides. The fusion proteins can be recombinantly produced at high expression levels. Also provided are related compositions and methods, including immunotherapeutic applications.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/053,530, filed Jan. 17, 2002, now pending, which claims the benefitof priority of U.S. Provisional Application No. 60/367,358 (formerlyU.S. Ser. No. 09/765,208, filed Jan. 17, 2001), the contents of whichare incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

The present invention relates generally to immunologically active,recombinant binding proteins, and in particular, to molecularlyengineered binding domain-immunoglobulin fusion proteins, includingsingle chain Fv-immunoglobulin fusion proteins. The present inventionalso relates to compositions and methods for treating malignantconditions and B-cell disorders, including diseases characterized byautoantibody production.

An immunoglobulin molecule is composed of two identical light chains andtwo identical heavy chains that are joined into a macromolecular complexby interchain disulfide bonds. Intrachain disulfide bonds join differentareas of the same polypeptide chain, which results in the formation ofloops that along with adjacent amino acids constitute the immunoglobulindomains. Each light chain and each heavy chain has a single variableregion that shows considerable variation in amino acid composition fromone antibody to another. The light chain variable region, V_(L),associates with the variable region of a heavy chain, V_(H), to form theantigen binding site of the immunoglobulin, Fv. Light chains have asingle constant region domain and heavy chains have several constantregion domains. Classes IgG, IgA, and IgD have three constant regiondomains, which are designated CH1, CH2, and CH3, and the IgM and IgEclasses have four constant region domains.

The heavy chains of immunoglobulins can be divided into three functionalregions: Fd, hinge, and Fc. The Fd region comprises the V_(H) and CH1domains and in combination with the light chain forms Fab. The Fcfragment is generally considered responsible for the effector functionsof an immunoglobulin, such as, complement fixation and binding to Fcreceptors. The hinge region, found in IgG, IgA, and IgD classes, acts asa flexible spacer, allowing the Fab portion to move freely in space. Incontrast to the constant regions, the hinge domains are structurallydiverse, varying in both sequence and length among immunoglobulinclasses and subclasses. For example, three human IgG subclasses, IgG1,IgG2, and IgG4, have hinge regions of 12-15 amino acids while IgG3comprises approximately 62 amino acids, including 21 proline residuesand 11 cysteine residues. According to crystallographic studies, thehinge can be further subdivided functionally into three regions: theupper hinge, the core, and the lower hinge (Shin et al., ImmunologicalReviews 130:87 (1992)). The upper hinge includes amino acids from thecarboxyl end of CH1 to the first residue in the hinge that restrictsmotion, generally the first cysteine residue that forms an interchaindisulfide bond between the two heavy chains. The length of the upperhinge region correlates with the segmental flexibility of the antibody.The core hinge region contains the inter-heavy chain disulfide bridges,and the lower hinge region joins the amino terminal end of the CH2domain and includes residues in CH2. (Id.) The core hinge region ofhuman IgG1 contains the sequence, Cys-Pro-Pro-Cys, which when disulfidebonds are formed results in a cyclic octa-peptide believed to act as apivot, thus conferring flexibility. The hinge region may also containcarbohydrate attachment sites. For example, IgA1 contains fivecarbohydrate sites within a 17 amino acid segment of the hinge region,conferring exception resistance of the hinge to intestinal proteases,considered an advantageous property for a secretory immunoglobulin.

Conformational changes permitted by the structure and flexibility of thehinge region may affect the effector functions of the Fc portion of theantibody. Three general categories of effector functions associated withthe Fc region include (1) activation of the classical complementcascade, (2) interaction with effector cells, and (3)compartmentalization of immunoglobulins. The different human IgGsubclasses vary in their relative efficacy to activate and amplify thesteps of the complement cascade. In general, IgG1 and IgG3 mosteffectively fix complement, IgG2 is less effective, and IgG4 does notactivate complement. Complement activation is initiated by binding ofC1q, a subunit of the first component C1 in the cascade, to anantigen-antibody complex. Even though the binding site for C1q islocated in the CH2 domain of the antibody, the hinge region influencesthe ability of the antibody to activate the cascade. For example,recombinant immunoglobulins lacking a hinge region are unable toactivate complement. (Id.) Without the flexibility conferred by thehinge region, the Fab portion of the antibody bound to the antigen maynot be able to adopt the conformation required to permit C1q to bind toCH2. (See id.) Studies have indicated that hinge length and segmentalflexibility correlate with complement activation; however, thecorrelation is not absolute. Human IgG3 molecules with altered hingeregions that are as rigid as IgG4 still effectively activate thecascade.

Lack of the hinge region also affects the ability of human IgGimmunoglobulins to bind Fc receptors on immune effector cells. Bindingof an immunoglobulin to an Fc receptor facilitates antibody-dependentcellular cytotoxicity (ADCC), which is presumed to be an important meansto eliminate tumor cells. The human IgG Fc receptor family is dividedinto three groups, FcγRI (CD64), which is capable of binding IgG withhigh affinity, FcγRII (CD32), and FcγRIII (CD16), both of which are lowaffinity receptors. The molecular interaction between each of the threereceptors and an immunoglobulin has not been defined precisely, butexperiments indicate that residues in the hinge proximal region of theCH2 domain are important to the specificity of the interaction betweenthe antibody and the Fc receptor. In addition, IgG1 myeloma proteins andrecombinant IgG3 chimeric antibodies that lack a hinge region are unableto bind FcγRI, likely because accessibility to CH2 is decreased. (Shinet al., Intern. Rev. Immunol. 10:177, 178-79 (1993)).

Monoclonal antibody technology and genetic engineering methods have ledto rapid development of immunoglobulin molecules for diagnosis andtreatment of human diseases. Protein engineering has been applied toimprove the affinity of an antibody for its cognate antigen, to diminishproblems related to immunogenicity, and to alter an antibody's effectorfunctions. The domain structure of immunoglobulins is amenable toengineering, in that the antigen binding domains and the domainsconferring effector functions may be exchanged between immunoglobulinclasses and subclasses.

In addition, smaller immunoglobulin molecules have been constructed toovercome problems associated with whole immunoglobulin therapy. Singlechain Fv (scFv) comprise the heavy chain variable domain joined via ashort linker peptide to the light chain variable domain (Huston et al.Proc. Natl. Acad. Sci. USA, 85: 5879-83, 1988). Because of the smallsize of scFv molecules, they exhibit very rapid clearance from plasmaand tissues and more effective penetration into tissues than wholeimmunoglobulin. An anti-tumor scFv showed more rapid tumor penetrationand more even distribution through the tumor mass than the correspondingchimeric antibody (Yokota et al., Cancer Res. 52, 3402-08 (1992)).Fusion of an scFv to another molecule, such as a toxin, takes advantageof the specific antigen-binding activity and the small size of an scFvto deliver the toxin to a target tissue. (Chaudary et al., Nature339:394 (1989); Batra et al., Mol. Cell. Biol. 11:2200 (1991)).

Despite the advantages that scFv molecules bring to serotherapy, severaldrawbacks to this therapeutic approach exist. While rapid clearance ofscFv may reduce toxic effects in normal cells, such rapid clearance mayprevent delivery of a minimum effective dose to the target tissue.Manufacturing adequate amounts of scFv for administration to patientshas been challenging due to difficulties in expression and isolation ofscFv that adversely affect the yield. During expression, scFv moleculeslack stability and often aggregate due to pairing of variable regionsfrom different molecules. Furthermore, production levels of scFvmolecules in mammalian expression systems are low, limiting thepotential for efficient manufacturing of scFv molecules for therapy(Davis et al, J. Biol. Chem. 265:10410-18 (1990); Traunecker et al.,EMBO J. 10: 3655-59 (1991)). Strategies for improving production havebeen explored, including addition of glycosylation sites to the variableregions (Jost, C. R. U.S. Pat. No. 5,888,773, Jost et al, J. Biol. Chem.269: 26267-73 (1994)).

Conjugation or fusion of toxins to scFV provides a very potent molecule,but dosing is limited by toxicity from the toxin molecule. Toxic effectsinclude elevation of liver enzymes and vascular leak syndrome. Inaddition, immunotoxins are highly immunogenic, and host antibodiesgenerated against the toxin limit its potential for repeated treatment.

An additional disadvantage to using scFv for therapy is the lack ofeffector function. An scFv without the cytolytic functions, ADCC andcomplement dependent-cytotoxicity (CDC), associated with the constantregion of an immunoglobulin may be ineffective for treating disease.Even though development of scFv technology began over 12 years ago,currently no scFv products are approved for therapy.

The benefit of antibody constant region-associated effector functions totreatment of a disease has prompted development of fusion proteins inwhich nonimmunoglobulin sequences are substituted for the antibodyvariable region. For example, CD4, the T cell surface protein recognizedby HIV, was recombinantly fused to an immunoglobulin Fc effector domain.(See Sensel et al., Chem. Immunol. 65:129-158 (1997)). The biologicalactivity of such a molecule will depend in part on the class or subclassof the constant region chosen. An IL-2-IgG1 fusion protein effectedcomplement-mediated lysis of IL-2 receptor-bearing cells. (See id.). Useof immunoglobulin constant regions to construct these and other fusionproteins may also confer improved pharmacokinetic properties.

Diseases and disorders thought to be amenable to some type ofimmunoglobulin therapy include cancer and immune system disorders.Cancer includes a broad range of diseases, affecting approximately onein four individuals worldwide. Rapid and unregulated proliferation ofmalignant cells is a hallmark of many types of cancer, includinghematological malignancies. Patients with a hematologic malignantcondition have benefited most from advances in cancer therapy in thepast two decades (Multani et al., J. Clin. Oncology 16: 3691-3710,1998). Although remission rates have increased, most patients stillrelapse and succumb to their disease. Barriers to cure with cytotoxicdrugs include tumor cell resistance and the high toxicity ofchemotherapy, which prevents optimal dosing in many patients. Newtreatments based on targeting with molecules that specifically bind to amalignant cell, including monoclonal antibodies (mAbs), can improveeffectiveness without increasing toxicity.

Since mAbs were first described in 1975 (Kohler et al., Nature256:495-97 (1975)), many patients have been treated with mAbs toantigens expressed on tumor cells. These studies have yielded importantlessons regarding the selection of target antigens suitable for therapy.First and most importantly, the target antigen should not be expressedby crucial normal tissues. Fortunately, hematologic malignant cellsexpress many antigens that are not expressed on stem cells or otheressential cells. Treatment of a hematologic malignant condition thatdepletes both normal and malignant cells of hematological origin hasbeen acceptable because regeneration of normal cells from progenitorsoccurs after therapy has ended. Second, the target antigen should beexpressed on all clonogenic populations of tumor cells, and expressionshould persist despite the selective pressure from immunoglobulintherapy. Thus, the choice of surface idiotype for therapy of B cellmalignancy has been limited by the outgrowth of tumor cell variants withaltered surface idiotype expression even though the antigen exhibits ahigh degree of tumor selectivity (Meeker et al., N. Engl. J. Med.312:1658-65 (1985)). Third, the selected antigen must traffic properlyafter an immunoglobulin binds to it. Shedding or internalization of atarget antigen after an immunoglobulin binds to the antigen may allowtumor cells to escape destruction, thus limiting the effectiveness ofserotherapy. Fourth, binding of an immunoglobulin to target antigensthat transmit activation signals may result in improved functionalresponses in tumor cells that lead to growth arrest and apoptosis. Whileall of these properties are important, the triggering of apoptosis afteran immunoglobulin binds to the antigen may be a critical factor inachieving successful serotherapy.

Antigens that have been tested as targets for serotherapy of B and Tcell malignancies include Ig idiotype (Brown et al., Blood 73:651-61(1989)), CD19 (Hekman et al., Cancer Immunol. Immunother. 32:364-72(1991); Vlasveld et al., Cancer Immunol. Immunother. 40: 37-47 (1995)),CD20 (Press et al., Blood 69: 584-91 (1987); Maloney et al., J. Clin.Oncol. 15:3266-74, (1997)) CD21 (Scheinberg et. al., J. Clin. Oncol.8:792-803, (1990)), CD5 (Dillman et. al., J. Biol. Respn. Mod. 5:394-410(1986)), and CD52 (CAMPATH) (Pawson et al., J. Clin. Oncol. 15:2667-72,(1997)). Of these, the most success has been obtained using CD20 as atarget for therapy of B cell lymphomas. Each of the other targets hasbeen limited by the biological properties of the antigen. For example,surface idiotype can be altered through somatic mutation, allowing tumorcell escape. CD5, CD21, and CD19 are rapidly internalized after mAbbinding, allowing tumor cells to escape destruction unless mAbs areconjugated with toxin molecules. CD22 is expressed on only a subset of Bcell lymphomas, while CD52 is expressed on both T cells and B cells andgenerates immunosuppression from T cell depletion.

CD20 fulfills the basic criteria described above for selection of anappropriate target antigen for therapy of a B cell malignant condition.Treatment of patients with low grade or follicular B cell lymphoma usingchimeric CD20 mAb induces partial or complete responses in many patients(McLaughlin et al, Blood 88:90a (abstract, suppl. 1) (1996); Maloney etal, Blood 90: 2188-95 (1997)). However, tumor relapse commonly occurswithin six months to one year. Therefore, further improvements inserotherapy are needed to induce more durable responses in low grade Bcell lymphoma, and to allow effective treatment of high grade lymphomaand other B cell diseases.

One approach to improving CD20 serotherapy has been to targetradioisotopes to B cell lymphomas using mAbs specific for CD20. Whilethe effectiveness of therapy is increased, associated toxicity from thelong in vivo half-life of the radioactive antibody increased also,sometimes requiring that the patient undergo stem cell rescue (Press etal., N. Eng. J. Med. 329: 1219-1224, 1993; Kaminski et al., N. Eng. J.Med. 329:459-65 (1993)). MAbs to CD20 have been cleaved with proteasesto yield F(ab′)₂ or Fab fragments prior to attachment of theradioisotope. This improves penetration of the radioisotope conjugateinto the tumor, and shortens the in vivo half-life, thus reducing thetoxicity to normal tissues. However, the advantages of effectorfunctions, including complement fixation and ADCC, that are provided bythe Fc region of the CD20 mAb are lost. Therefore, for improved deliveryof radioisotopes, a strategy is needed to make a CD20 mAb derivativethat retains Fc-dependent effector functions but which is smaller insize, thereby increasing tumor penetration and shortening mAb half-life.

CD20 was the first human B cell lineage-specific surface moleculeidentified by a monoclonal antibody, but the function of CD20 in B cellbiology is still incompletely understood. CD20 is a non-glycosylated,hydrophobic 35 kDa phosphoprotein that has both amino and carboxy endsin the cytoplasm (Einfeld et al, EMBO J. 7:711-17 (1988)). Naturalligands for CD20 have not been identified. CD20 is expressed by allnormal mature B cells, but is not expressed by precursor B cells.

CD20 mAbs deliver signals to normal B cells that affect viability andgrowth (Clark et al., Proc. Natl. Acad. Sci. USA 83:4494-98 (1986)).Recent data has shown that extensive cross-linking of CD20 can induceapoptosis of B lymphoma cell lines (Shan et al., Blood 91:1644-52(1998)). Cross-linking of CD20 on the cell surface increases themagnitude and kinetics of signal transduction, which was detected bymeasuring phosphorylation of cellular substrates on tyrosine residues(Deans et al., J. Immunol. 146:846-53 (1993)). Importantly, apoptosis ofRamos B lymphoma cells was also be induced by cross-linking of CD20 mAbsby addition of Fc-receptor positive cells (Shan et al., Blood 91:1644-52 (1998)). Therefore, in addition to cellular depletion bycomplement and ADCC mechanisms, Fc-receptor binding by CD20 mAbs in vivocould promote apoptosis of malignant B cells by CD20 cross-linking. Thistheory is consistent with experiments showing that effectiveness of CD20therapy of human lymphoma in a SCID mouse model was dependent uponFc-receptor binding by the CD20 mAb (Funakoshi et al., J. Immunotherapy19:93-101 (1996)).

The CD20 polypeptide contains four transmembrane domains (Einfeld etal., EMBO J. 7: 711-17, (1988); Stamenkovic et al., J. Exp. Med.167:1975-80 (1988); Tedder et. al., J. Immunol. 141:4388-4394 (1988)).The multiple membrane spanning domains prevent CD20 internalizationafter antibody binding. This property of CD20 was recognized as animportant feature for effective therapy of B cell malignancies when amurine CD20 mAb, 1F5, was injected into patients with B cell lymphoma,resulting in significant depletion of malignant cells and partialclinical responses (Press et al., Blood 69: 584-91 (1987)).

Because normal mature B cells also express CD20, normal B cells aredepleted during CD20 antibody therapy (Reff, M. E. et al, Blood 83:435-445, 1994). However, after treatment is completed, normal B cellsare regenerated from CD20 negative B cell precursors; therefore,patients treated with anti-CD20 therapy do not experience significantimmunosuppression. Depletion of normal B cells may be beneficial indiseases that involve inappropriate production of autoantibodies orother diseases where B cells may play a role. A chimeric mAb specificfor CD20, consisting of heavy and light chain variable regions of mouseorigin fused to human IgG1 heavy chain and human kappa light chainconstant regions, retained binding to CD20 and the ability to mediateADCC and to fix complement (Liu et al., J. Immunol. 139:3521-26 (1987);Robinson et al., U.S. Pat. No. 5,500,362). This work led to developmentof a chimeric CD20 mAb, Rituximab™, currently approved by the U.S. Foodand Drug Administration for approval for therapy of B cell lymphomas.While clinical responses are frequently observed after treatment withRituximab™, patients often relapse after about 6-12 months.

High doses of Rituximab™ are required for intravenous injection becausethe molecule is large, approximately 150 kDa, and diffusion is limitedinto the lymphoid tissues where many tumor cells reside. The mechanismof anti-tumor activity of Rituximab™ is thought to be a combination ofseveral activities, including ADCC, fixation of complement, andtriggering of signals in malignant B cells that promote apoptosis. Thelarge size of Rituximab™ prevents optimal diffusion of the molecule intolymphoid tissues that contain malignant B cells, thereby limiting theseanti-tumor activities. As discussed above, cleavage of CD20 mAbs withproteases into Fab or F(ab′)₂ fragments makes them smaller and allowsbetter penetration into lymphoid tissues, but the effector functionsimportant for anti-tumor activity are lost. While CD20 mAb fragments maybe more effective than intact antibody for delivery of radioisotopes, itwould be desirable to construct a CD20 mAb derivative that retains theeffector functions of the Fc portion, but is smaller in size,facilitating better tumor penetration and resulting in a shorterhalf-life.

CD20 is expressed by malignant cells of B cell origin, including B celllymphoma and chronic lymphocytic leukemia (CLL). CD20 is not expressedby malignancies of pre-B cells, such as acute lymphoblastic leukemia.CD20 is therefore a good target for therapy of B cell lymphoma, CLL, andother diseases in which B cells are involved in the disease activity.Other B cell disorders include autoimmune diseases in whichautoantibodies are produced during the differentiation of B cells intoplasma cells. Examples of B cell disorders include autoimmune thyroiddisease, including Graves' disease and Hashimoto's thyroiditis,rheumatoid arthritis, systemic lupus erythematosus (SLE), Sjogrenssyndrome, immune thrombocytopenic purpura (ITP), multiple sclerosis(MS), myasthenia gravis (MG), psoriasis, scleroderma, and inflammatorybowel disease, including Crohn's disease and ulcerative colitis.

From the foregoing, a clear need is apparent for improved compositionsand methods to treat malignant conditions and B cell disorders. Thecompositions and methods of the present invention overcome thelimitations of the prior art by providing a bindingdomain-immunoglobulin fusion protein comprising a binding domainpolypeptide that is fused to an immunoglobulin hinge region polypeptide,which is fused to an immunoglobulin heavy chain CH2 constant regionpolypeptide fused to an immunoglobulin heavy chain CH3 constant regionpolypeptide, wherein the binding domain-immunoglobulin fusion protein iscapable of mediating ADCC or complement fixation. Furthermore, thecompositions and methods offer other related advantages.

SUMMARY OF THE INVENTION

It is an aspect of the present invention to provide a bindingdomain-immunoglobulin fusion protein, comprising (a) a binding domainpolypeptide that is fused to an immunoglobulin hinge region polypeptide,wherein said hinge region polypeptide is selected from the groupconsisting of (i) a mutated hinge region polypeptide that contains nocysteine residues and that is derived from a wild-type immunoglobulinhinge region polypeptide having one or more cysteine residues, (ii) amutated hinge region polypeptide that contains one cysteine residue andthat is derived from a wild-type immunoglobulin hinge region polypeptidehaving two or more cysteine residues, (iii) a wild-type human IgA hingeregion polypeptide, (iv) a mutated human IgA hinge region polypeptidethat contains no cysteine residues and that is derived from a wild-typehuman IgA region polypeptide, and (v) a mutated human IgA hinge regionpolypeptide that contains one cysteine residue and that is derived froma wild-type human IgA region polypeptide; (b) an immunoglobulin heavychain CH2 constant region polypeptide that is fused to the hinge regionpolypeptide; and (c) an immunoglobulin heavy chain CH3 constant regionpolypeptide that is fused to the CH2 constant region polypeptide,wherein: (1) the binding domain-immunoglobulin fusion protein is capableof at least one immunological activity selected from the groupconsisting of antibody dependent cell-mediated cytotoxicity andcomplement fixation, and (2) the binding domain polypeptide is capableof specifically binding to an antigen. In one embodiment theimmunoglobulin hinge region polypeptide is a mutated hinge regionpolypeptide and exhibits a reduced ability to dimerize, relative to awild-type human immunoglobulin G hinge region polypeptide. In anotherembodiment the binding domain polypeptide comprises at least oneimmunoglobulin variable region polypeptide that is an immunoglobulinlight chain variable region polypeptide or an immunoglobulin heavy chainvariable region polypeptide. In a further embodiment the immunoglobulinvariable region polypeptide is derived from a human immunoglobulin.

In another embodiment the binding domain Fv-immunoglobulin fusionprotein binding domain polypeptide comprises (a) at least oneimmunoglobulin light chain variable region polypeptide; (b) at least oneimmunoglobulin heavy chain variable region polypeptide; and (c) at leastone linker peptide that is fused to the polypeptide of (a) and to thepolypeptide of (b). In a further embodiment the immunoglobulin lightchain variable region and heavy chain variable region polypeptides arederived from human immunoglobulins.

In another embodiment at least one of the immunoglobulin heavy chain CH2constant region polypeptide and the immunoglobulin heavy chain CH3constant region polypeptide is derived from a human immunoglobulin heavychain. In another embodiment the immunoglobulin heavy chain constantregion CH2 and CH3 polypeptides are of an isotype selected from humanIgG and human IgA. In another embodiment the antigen is selected fromthe group consisting of CD19, CD20, CD37, CD40 and L6. In certainfurther embodiments of the above described fusion protein, the linkerpolypeptide comprises at least one polypeptide having as an amino acidsequence Gly-Gly-Gly-Gly-Ser, and in certain other embodiments thelinker polypeptide comprises at least three repeats of a polypeptidehaving as an amino acid sequence Gly-Gly-Gly-Gly-Ser. In certainembodiments the immunoglobulin hinge region polypeptide comprises ahuman IgA hinge region polypeptide. In certain embodiments the bindingdomain polypeptide comprises a CD154 extracellular domain. In certainembodiments the binding domain polypeptide comprises a CD154extracellular domain and at least one immunoglobulin variable regionpolypeptide.

In other embodiments the invention provides an isolated polynucleotideencoding any of the above described binding domain-immunoglobulin fusionproteins, and in related embodiments the invention provides arecombinant expression construct comprising such a polynucleotide, andin certain further embodiments the invention provides a host celltransformed or transfected with such a recombinant expression construct.In another embodiment the invention provides a method of producing abinding domain-immunoglobulin fusion protein, comprising the steps of(a) culturing the host cell as just described, under conditions thatpermit expression of the binding domain-immunoglobulin fusion protein;and (b) isolating the binding domain-immunoglobulin fusion protein fromthe host cell culture.

The present invention also provides in certain embodiments apharmaceutical composition comprising a binding domain-immunoglobulinfusion protein as described above, in combination with a physiologicallyacceptable carrier. In another embodiment there is provided a method oftreating a subject having or suspected of having a malignant conditionor a B-cell disorder, comprising administering to a patient atherapeutically effective amount of an above described bindingdomain-immunoglobulin fusion protein. In certain further embodiments themalignant condition or B-cell disorder is a B-cell lymphoma or a diseasecharacterized by autoantibody production, and in certain other furtherembodiments the malignant condition or B-cell disorder is rheumatoidarthritis, myasthenia gravis, Grave's disease, type I diabetes mellitus,multiple sclerosis or an autoimmune disease.

These and other aspects of the present invention will become apparentupon reference to the following detailed description and attacheddrawings. All references disclosed herein are hereby incorporated byreference in their entireties as if each was incorporated individually.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B show DNA and deduced amino acid sequences (SEQ ID NO:15) of2H7scFv-Ig, a binding domain-immunoglobulin fusion protein capable ofspecifically binding CD20.

FIG. 2 shows production levels of 2H7 scFv-Ig by transfected, stable CHOlines and generation of a standard curve by binding of purified 2H7scFv-Ig to CHO cells expressing CD20.

FIG. 3 shows SDS-PAGE analysis of multiple preparations of isolated2H7scFv-Ig protein.

FIG. 4 shows complement fixation (FIG. 4A) and mediation ofantibody-dependent cellular cytotoxicity (ADCC, FIG. 4B)) by 2H7scFv-Ig.

FIG. 5 shows the effect of simultaneous ligation of CD20 and CD40 ongrowth of normal B cells.

FIGS. 6A-6B show the effect of simultaneous ligation of CD20 and CD40 onCD95 expression (6A) and induction of apoptosis in a B lymphoblastoidcell line (6B).

FIG. 7 shows DNA and deduced amino acid sequences of 2H7scFv-CD154 L2(FIG. 7A, SEQ ID NOS:21 and 33) and 2H7scFv-CD154 S4 (FIG. 7B, SEQ IDNOS:22 and 34) binding domain-immunoglobulin fusion proteins capable ofspecifically binding CD20 and CD40.

FIG. 8 shows binding of 2H7scFv-CD154 binding domain-immunoglobulinfusion proteins to CD20+ CHO cells by flow immunocytofluorimetry.

FIG. 9 shows binding of Annexin V to B cell lines Ramos, BJAB, and T51after binding of 2H7scFv-CD154 binding domain-immunoglobulin fusionprotein to cells.

FIG. 10 shows effects on proliferation of B cell line T51 followingbinding of 2H7scFv-CD154 binding domain-immunoglobulin fusion protein.

FIG. 11 depicts schematic representations of the structures of2H7ScFv-Ig fusion proteins (SEQ ID NOS:17, 16, AND 18) referred to asCytoxB or CytoxB derivatives: CytoxB-MHWTG1C (2H7 ScFv, mutant hinge,wild-type human IgG1 Fc domain), CytoxB-MHMG1C (2H7 ScFv, mutant hinge,mutated human IgG1 Fc domain) and CytoxB-IgAHWTHG1C (2H7 ScFv, humanIgA-derived hinge, wild-type human IgG1 Fc domain) respectively. Arrowsindicate position numbers of amino acid residues believed to contributeto FcR binding and ADCC activity (heavy arrows), and to complementfixation (light arrows). Note absence of interchain disulfide bonds.

FIG. 12 shows SDS-PAGE analysis of isolated CytoxB and 2H7scFv-CD154binding domain-immunoglobulin fusion proteins.

FIG. 13 shows antibody dependent cell-mediated cytotoxicity (ADCC)activity of CytoxB derivatives.

FIG. 14 shows complement dependent cytotoxicity (CDC) of CytoxBderivatives.

FIG. 15 shows serum half-life determinations of CytoxB-MHWTG1C inmacaque blood samples.

FIG. 16 shows effects of CytoxB-MHWTG1C on levels of circulating CD40+ Bcells in macaque blood samples.

FIG. 17 shows production levels of HD37 (CD19-specific) ScFv-Ig bytransfected mammalian cell lines and generation of a standard curve bybinding of purified HD37 ScFv-Ig to cells expressing CD19.

FIG. 18 shows production levels of L6 (carcinoma antigen) ScFv-Ig bytransfected, stable CHO lines and generation of a standard curve bybinding of purified L6 ScFv-Ig to cells expressing L6 antigen.

FIGS. 19A-19C show ADCC activity of binding domain-immunoglobulin fusionproteins 2H7 ScFv-Ig (19A), HD37 ScFv-Ig (19C) and G28-1 (CD37-specific)ScFv-Ig (19B).

FIG. 20 shows ADCC activity of L6 ScFv-Ig fusion proteins.

FIG. 21 shows SDS-PAGE analysis of L6 ScFv-Ig and 2H7 ScFv-Ig fusionproteins.

FIG. 22 shows SDS-PAGE analysis of G28-1 ScFv-Ig and HD37 ScFv-Ig fusionproteins.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to binding domain-immunoglobulinfusion proteins and to related compositions and methods, which will beuseful in immunotherapeutic and immunodiagnostic applications, and whichoffer certain advantages over antigen-specific polypeptides of the priorart. The fusion proteins of the present invention are preferably singlepolypeptide chains that comprise, in pertinent part, the following fuseddomains: a binding domain polypeptide, an immunoglobulin hinge regionpolypeptide, an immunoglobulin heavy chain CH2 constant regionpolypeptide, and an immunoglobulin heavy chain CH3 constant regionpolypeptide. In particularly preferred embodiments, the polypeptidedomains of which the binding domain-immunoglobulin fusion protein iscomprised are, or are derived from, polypeptides that are the productsof human gene sequences, but the invention need not be so limited andmay in fact relate to binding domain-immunoglobulin fusion proteins asprovided herein that are derived from any natural or artificial source,including genetically engineered and/or mutated polypeptides.

The present invention relates in part to the surprising observation thatthe binding domain-immunoglobulin fusion proteins described herein arecapable of immunological activity. More specifically, these proteinsretain the ability to participate in well known immunological effectoractivities including antibody dependent cell mediated cytotoxicity(ADCC, e.g., subsequent to antigen binding on a cell surface, engagementand induction of cytotoxic effector cells bearing appropriate Fcreceptors, such as natural killer (NK) cells bearing FcRγIII, underappropriate conditions) and/or complement fixation in complementdependent cytotoxicity (CDC, e.g., subsequent to antigen binding on acell surface, recruitment and activation of cytolytic proteins that arecomponents of the blood complement cascade), despite having structuresthat would not be expected to be capable of promoting such effectoractivities. As described in greater detail below, ADCC and CDC areunexpected functions for monomeric proteins comprising immunoglobulinheavy chain regions, which are favored by the structures selected forthe subject fusion proteins, and particularly by the selection of hingeregion polypeptides that are compromised in their ability to forminterchain, homodimeric disulfide bonds.

Another advantage afforded by the present invention is a bindingdomain-immunoglobulin fusion polypeptide that can be produced insubstantial quantities that are typically greater than those routinelyattained with single-chain antibody constructs of the prior art. Inpreferred embodiments, the binding domain-immunoglobulin fusionpolypeptides of the present invention are recombinantly expressed inmammalian expression systems, which offer the advantage of providingpolypeptides that are stable in vivo (e.g., under physiologicalconditions). According to non-limiting theory, such stability may derivein part from posttranslational modifications, and specificallyglycosylation, of the fusion proteins. Production of the present bindingdomain-immunoglobulin fusion proteins via recombinant mammalianexpression has been attained in static cell cultures at a level ofgreater than 50 mg protein per liter culture supernatant and has beenroutinely observed in such cultures at 10-50 mg/l, such that preferablyat least 10-50 mg/l may be produced under static culture conditions;also contemplated are enhanced production of the fusion proteins usingart-accepted scale-up methodologies such as “fed batch” (i.e.,non-static) production, where yields of at least 5-500 mg/l, and in someinstances at least 0.5-1 gm/l, depending on the particular proteinproduct, are obtained.

A binding domain polypeptide according to the present invention may beany polypeptide that possesses the ability to specifically recognize andbind to a cognate biological molecule or complex of more than onemolecule or assembly or aggregate, whether stable or transient, of sucha molecule, which includes a protein, polypeptide, peptide, amino acid,or derivative thereof; a lipid, fatty acid or the like, or derivativethereof; a carbohydrate, saccharide or the like or derivative thereof, anucleic acid, nucleotide, nucleoside, purine, pyrimidine or relatedmolecule, or derivative thereof, or the like; or any combination thereofsuch as, for example, a glycoprotein, a glycopeptide, a glycolipid, alipoprotein, a proteolipid; or any other biological molecule that may bepresent in a biological sample. Biological samples may be provided byobtaining a blood sample, biopsy specimen, tissue explant, organculture, biological fluid or any other tissue or cell preparation from asubject or a biological source. The subject or biological source may bea human or non-human animal, a primary cell culture or culture adaptedcell line including but not limited to genetically engineered cell linesthat may contain chromosomally integrated or episomal recombinantnucleic acid sequences, immortalized or immortalizable cell lines,somatic cell hybrid cell lines, differentiated or differentiatable celllines, transformed cell lines and the like. In certain preferredembodiments of the invention, the subject or biological source may besuspected of having or being at risk for having a malignant condition ora B-cell disorder as provided herein, which in certain further preferredembodiments may be an autoimmune disease, and in certain other preferredembodiments of the invention the subject or biological source may beknown to be free of a risk or presence of such disease.

A binding domain polypeptide may therefore be any naturally occurring orrecombinantly produced binding partner for a cognate biological moleculeas provided herein that is a target structure of interest, hereinreferred to as an “antigen” but intended according to the presentdisclosure to encompass any target biological molecule to which it isdesirable to have the subject invention fusion protein specificallybind. Binding domain-immunoglobulin fusion proteins are defined to be“immunospecific” or capable of specifically binding if they bind adesired target molecule such as an antigen as provided herein, with aK_(a) of greater than or equal to about 10⁴ M⁻¹, preferably of greaterthan or equal to about 10⁵ M⁻¹, more preferably of greater than or equalto about 10⁶ M⁻¹ and still more preferably of greater than or equal toabout 10⁷ M⁻¹. Affinities of binding domain-immunoglobulin fusionproteins according to the present invention can be readily determinedusing conventional techniques, for example those described by Scatchardet al., Ann. N.Y. Acad. Sci. 51:660 (1949). Such determination of fusionprotein binding to target antigens of interest can also be performedusing any of a number of known methods for identifying and obtainingproteins that specifically interact with other proteins or polypeptides,for example, a yeast two-hybrid screening system such as that describedin U.S. Pat. No. 5,283,173 and U.S. Pat. No. 5,468,614, or theequivalent.

Preferred embodiments of the subject invention bindingdomain-immunoglobulin fusion protein comprise binding domains thatinclude at least one immunoglobulin variable region polypeptide, such asall or a portion or fragment of a heavy chain or a light chain V-region,provided it is capable of specifically binding an antigen or otherdesired target structure of interest as described herein. In otherpreferred embodiments the binding domain comprises a single chainimmunoglobulin-derived Fv product, which may include all or a portion ofat least one immunoglobulin light chain V-region and all or a portion ofat least one immunoglobulin heavy chain V-region, and which furthercomprises a linker fused to the V-regions; preparation and testing suchconstructs are described in greater detail herein and are well known inthe art. Other binding domain polypeptides may comprise any protein orportion thereof that retains the ability to specifically bind an antigenas provided herein, including non-immunoglobulins. Accordingly theinvention contemplates fusion proteins comprising binding domainpolypeptides that are derived from polypeptide ligands such as hormones,cytokines, chemokines, and the like; cell surface or soluble receptorsfor such polypeptide ligands; lectins; intercellular adhesion receptorssuch as specific leukocyte integrins, selectins, immunoglobulin genesuperfamily members, intercellular adhesion molecules (ICAM-1, -2, -3)and the like; histocompatibility antigens; etc.

Examples of cell surface receptors that may provide a binding domainpolypeptide, and that may also be selected as the target molecule orantigen to which a binding domain-Ig fusion protein of the presentinvention desirably binds, include the following, or the like: HER1(e.g., GenBank Accession Nos. U48722, SEG_HEGFREXS, KO3193), HER2(Yoshino et al., 1994 J. Immunol. 152:2393; Disis et al., 1994 Canc.Res. 54:16; see also, e.g., GenBank Acc. Nos. X03363, M17730,SEG_HUMHER20), HER3 (e.g., GenBank Acc. Nos. U29339, M34309), HER4(Plowman et al., 1993 Nature 366:473; see also e.g., GenBank Acc. Nos.L07868, T64105), epidermal growth factor receptor (EGFR) (e.g., GenBankAcc. Nos. U48722, SEG_HEGFREXS, KO3193), vascular endothelial cellgrowth factor (e.g., GenBank No. M32977), vascular endothelial cellgrowth factor receptor (e.g., GenBank Acc. Nos. AF022375, 1680143,U48801, X62568), insulin-like growth factor-I (e.g., GenBank Acc. Nos.X00173, X56774, X56773, X06043, see also European Patent No. GB2241703), insulin-like growth factor-II (e.g., GenBank Acc. Nos. X03562,X00910, SEG_HUMGFIA, SEG_HUMGFI2, M17863, M17862), transferrin receptor(Trowbridge and Omary, 1981 Proc. Nat. Acad. USA 78:3039; see also e.g.,GenBank Acc. Nos. X01060, M11507), estrogen receptor (e.g., GenBank Acc.Nos. M38651, X03635, X99101, U47678, M12674), progesterone receptor(e.g., GenBank Acc. Nos. X51730, X69068, M15716), follicle stimulatinghormone receptor (FSH-R) (e.g., GenBank Acc. Nos. Z34260, M65085),retinoic acid receptor (e.g., GenBank Acc. Nos. L12060, M60909, X77664,X57280, X07282, X06538), MUC-1 (Barnes et al., 1989 Proc. Nat. Acad.Sci. USA 86:7159; see also e.g., GenBank Acc. Nos. SEG_MUSMUCIO, M65132,M64928) NY-ESO-1 (e.g., GenBank Acc. Nos. AJ003149, U87459), NA 17-A(e.g., European Patent No. WO 96/40039), Melan-A/MART-1 (Kawakami etal., 1994 Proc. Nat. Acad. Sci. USA 91:3515; see also e.g., GenBank Acc.Nos. U06654, U06452), tyrosinase (Topalian et al., 1994 Proc. Nat. Acad.Sci. USA 91:9461; see also e.g., GenBank Acc. Nos. M26729, SEG_HUMTYR0,see also Weber et al., J. Clin. Invest (1998) 102:1258), Gp-100(Kawakami et al., 1994 Proc. Nat. Acad. Sci. USA 91:3515; see also e.g.,GenBank Acc. No. S73003, see also European Patent No. EP 668350; Ademaet al., 1994 J. Biol. Chem. 269:20126), MAGE (van den Bruggen et al.,1991 Science 254:1643; see also e.g, GenBank Acc. Nos. U93163, AF064589,U66083, D32077, D32076, D32075, U10694, U10693, U10691, U10690, U10689,U10688, U10687, U10686, U10685, L18877, U10340, U10339, L18920, U03735,M77481), BAGE (e.g., GenBank Acc. No. U19180, see also U.S. Pat. Nos.5,683,886 and 5,571,711), GAGE (e.g., GenBank Acc. Nos. AF055475,AF055474, AF055473, U19147, U19146, U19145, U19144, U19143, U19142), anyof the CTA class of receptors including in particular HOM-MEL-40 antigenencoded by the SSX2 gene (e.g., GenBank Acc. Nos. X86175, U90842,U90841, X86174), carcinoembyonic antigen (CEA, Gold and Freedman, 1985J. Exp. Med. 121:439; see also e.g., GenBank Acc. Nos. SEG_HUMCEA,M59710, M59255, M29540), and PyLT (e.g., GenBank Acc. Nos. J02289,J02038).

Additional cell surface receptors that may be sources of binding domainpolypeptides or that may be cognate antigens include the following, orthe like: CD2 (e.g., GenBank Acc. Nos. Y00023, SEG_HUMCD2, M16336,M16445, SEG_MUSCD2, M14362), 4-1BB (CDw137, Kwon et al., 1989 Proc. Nat.Acad. Sci. USA 86:1963, 4-1BB ligand (Goodwin et al., 1993 Eur. J.Immunol. 23:2361; Melero et al., 1998 Eur. J. Immunol. 3:116), CD5(e.g., GenBank Acc. Nos. X78985, X89405), CD10 (e.g., GenBank Acc. Nos.M81591, X76732) CD27 (e.g., GenBank Acc. Nos. M63928, L24495, L08096),CD28 (June et al., 1990 Immunol. Today 11:211; see also, e.g., GenBankAcc. Nos. J02988, SEG_HUMCD28, M34563), CTLA-4 (e.g., GenBank Acc. Nos.L15006, X05719, SEG_HUMIGCTL), CD40 (e.g., GenBank Acc. Nos. M83312,SEG_MUSCO40A0, Y10507, X67878, X96710, U15637, L07414), interferon-γ(IFN-γ; see, e.g., Farrar et al. 1993 Ann. Rev. Immunol. 11:571 andreferences cited therein, Gray et al. 1982 Nature 295:503, Rinderknechtet al. 1984 J. Biol. Chem. 259:6790, DeGrado et al. 1982 Nature300:379), interleukin-4 (IL-4; see, e.g., 53^(rd) Forum in Immunology,1993 Research in Immunol. 144:553-643; Banchereau et al., 1994 in TheCytokine Handbook, 2^(nd) ed., A. Thomson, ed., Academic Press, NY, p.99; Keegan et al., 1994 J Leukocyt. Biol. 55:272, and references citedtherein), interleukin-17 (IL-17) (e.g., GenBank Acc. Nos. U32659,U43088) and interleukin-17 receptor (IL-17R) (e.g., GenBank Acc. Nos.U31993, U58917). Notwithstanding the foregoing, the present inventionexpressly does not encompass any immunoglobulin fusion protein that isdisclosed in U.S. Pat. No. 5,807,734, U.S. Pat. No. 5,795,572 or U.S.Pat. No. 5,807,734.

Additional cell surface receptors that may be sources of binding domainpolypeptides or that may be cognate antigens include the following, orthe like: CD59 (e.g., GenBank Acc. Nos. SEG_HUMCD590, M95708, M34671),CD48 (e.g., GenBank Acc. Nos. M59904), CD58/LFA-3 (e.g., GenBank Acc.No. A25933, Y00636, E12817; see also JP 1997075090-A), CD72 (e.g.,GenBank Acc. Nos. AA311036, S40777, L35772), CD70 (e.g., GenBank Acc.Nos. Y13636, S69339), CD80/B7.1 (Freeman et al., 1989 J. Immunol.43:2714; Freeman et al., 1991 J. Exp. Med. 174:625; see also e.g.,GenBank Acc. Nos. U33208, I683379), CD86/B7.2 (Freeman et al., 1993 J.Exp. Med. 178:2185, Boriello et al., 1995 J. Immunol. 155:5490; seealso, e.g., GenBank Acc. Nos. AF099105, SEG_MMB72G, U39466, U04343,SEG_HSB725, L25606, L25259), CD40 ligand (e.g., GenBank Acc. Nos.SEG_HUMCD40L, X67878, X65453, L07414), IL-17 (e.g., GenBank Acc. Nos.U32659, U43088), CD43 (e.g., GenBank Acc. Nos. X52075, J04536) and VLA-4(α₄β₇) (e.g., GenBank Acc. Nos. L12002, X16983, L20788, U97031, L24913,M68892, M95632). The following cell surface receptors are typicallyassociated with B cells: CD19 (e.g., GenBank Acc. Nos. SEG_HUMCD19W0,M84371, SEG_MUSCD19W, M62542), CD20 (e.g., GenBank Acc. Nos.SEG_HUMCD20, M62541), CD22 (e.g., GenBank Acc. Nos. 1680629, Y10210,X59350, U62631, X52782, L16928), CD30 ligand (e.g., GenBank Acc. Nos.L09753, M83554), CD37 (e.g., GenBank Acc. Nos. SEG_MMCD37X, X14046,X53517), CD106 (VCAM-1) (e.g., GenBank Acc. Nos. X53051, X67783,SEG_MMVCAM1C, see also U.S. Pat. No. 5,596,090), CD54 (ICAM-1) (e.g.,GenBank Acc. Nos. X84737, S82847, X06990, J03132, SEG_MUSICAM0),interleukin-12 (see, e.g., Reiter et al, 1993 Crit. Rev. Immunol. 13:1,and references cited therein). Accessory cell agents may also includeany of the following cell surface receptors typically associated withdendritic cells: CD83 (e.g., GenBank Acc. Nos. AF001036, AL021918),DEC-205 (e.g., GenBank Acc. Nos. AF011333, U19271).

An immunoglobulin hinge region polypeptide, as discussed above, includesany hinge peptide or polypeptide that occurs naturally, as an artificialpeptide or as the result of genetic engineering and that is situated inan immunoglobulin heavy chain polypeptide between the amino acidresidues responsible for forming intrachain immunoglobulin-domaindisulfide bonds in CH1 and CH2 regions; hinge region polypeptides foruse in the present invention may also include a mutated hinge regionpolypeptide. Accordingly, an immunoglobulin hinge region polypeptide maybe derived from, or may be a portion or fragment of (i.e., one or moreamino acids in peptide linkage, typically 5-65 amino acids, preferably10-50, more preferably 15-35, still more preferably 18-32, still morepreferably 20-30, still more preferably 21, 22, 23, 24, 25, 26, 27, 28or 29 amino acids) an immunoglobulin polypeptide chain regionclassically regarded as having hinge function, as described above, but ahinge region polypeptide for use in the instant invention need not be sorestricted and may include amino acids situated (according to structuralcriteria for assigning a particular residue to a particular domain thatmay vary, as known in the art) in an adjoining immunoglobulin domainsuch as a CH1 domain or a CH2 domain, or in the case of certainartificially engineered immunoglobulin constructs, an immunoglobulinvariable region domain.

Wild-type immunoglobulin hinge region polypeptides include any naturallyoccurring hinge region that is located between the constant regiondomains, CH1 and CH2, of an immunoglobulin. The wild-type immunoglobulinhinge region polypeptide is preferably a human immunoglobulin hingeregion polypeptide, preferably comprising a hinge region from a humanIgG immunoglobulin, and more preferably, a hinge region polypeptide froma human IgG1 isotype. As is known to the art, despite the tremendousoverall diversity in immunoglobulin amino acid sequences, immunoglobulinprimary structure exhibits a high degree of sequence conservation inparticular portions of immunoglobulin polypeptide chains, notably withregard to the occurrence of cysteine residues which, by virtue of theirsulfyhydryl groups, offer the potential for disulfide bond formationwith other available sulfydryl groups. Accordingly, in the context ofthe present invention wild-type immunoglobulin hinge region polypeptidesmay be regarded as those that feature one or more highly conserved(e.g., prevalent in a population in a statistically significant manner)cysteine residues, and in certain preferred embodiments a mutated hingeregion polypeptide may be selected that contains zero or one cysteineresidue and that is derived from such a wild-type hinge region.

A mutated immunoglobulin hinge region polypeptide may comprise a hingeregion that has its origin in an immunoglobulin of a species, of animmunoglobulin isotype or class, or of an immunoglobulin subclass thatis different from that of the CH2 and CH3 domains. For instance, incertain embodiments of the invention, the binding domain-immunoglobulinfusion protein may comprise a binding domain polypeptide that is fusedto an immunoglobulin hinge region polypeptide comprising a wild-typehuman IgA hinge region polypeptide, or a mutated human IgA hinge regionpolypeptide that contains zero or only one cysteine residues, asdescribed herein. Such a hinge region polypeptide may be fused to animmunoglobulin heavy chain CH2 region polypeptide from a different Igisotype or class, for example an IgG subclass, which in certainpreferred embodiments will be the IgG1 subclass.

For example, and as described in greater detail below, in certainembodiments of the present invention an immunoglobulin hinge regionpolypeptide is selected which is derived from a wild-type human IgAhinge region that naturally comprises three cysteines, where theselected hinge region polypeptide is truncated relative to the completehinge region such that only one of the cysteine residues remains (e.g.,SEQ ID NO:36). Similarly, in certain other embodiments of the invention,the binding domain-immunoglobulin fusion protein comprises a bindingdomain polypeptide that is fused to an immunoglobulin hinge regionpolypeptide comprising a mutated hinge region polypeptide in which thenumber of cysteine residues is reduced by amino acid substitution ordeletion. A mutated hinge region polypeptide may thus be derived from awild-type immunoglobulin hinge region that contains one or more cysteineresidues. In certain embodiments, a mutated hinge region polypeptide maycontain zero or only one cysteine residue, wherein the mutated hingeregion polypeptide is derived from a wild type immunoglobulin hingeregion that contains, respectively, one or more or two or more cysteineresidues. In the mutated hinge region polypeptide, the cysteine residuesof the wild-type immunoglobulin hinge region are preferably substitutedwith amino acids that are incapable of forming a disulfide bond. In oneembodiment of the invention, the mutated hinge region polypeptide isderived from a human IgG wild-type hinge region polypeptide, which mayinclude any of the four human IgG isotype subclasses, IgG1, IgG2, IgG3or IgG4. In certain preferred embodiments, the mutated hinge regionpolypeptide is derived from a human IgG1 wild-type hinge regionpolypeptide. By way of example, a mutated hinge region polypeptidederived from a human IgG1 wild-type hinge region polypeptide maycomprise mutations at two of the three cysteine residues in thewild-type immunoglobulin hinge region, or mutations at all threecysteine residues.

The cysteine residues that are present in a wild-type immunoglobulinhinge region and that are removed by mutagenesis according toparticularly preferred embodiments of the present invention includecysteine residues that form, or that are capable of forming, interchaindisulfide bonds. Without wishing to be bound by theory, the presentinvention contemplates that mutation of such hinge region cysteineresidues, which are believed to be involved in formation of interchaindisulfide bridges, reduces the ability of the subject invention bindingdomain-immunoglobulin fusion protein to dimerize (or form higheroligomers) via interchain disulfide bond formation, while surprisinglynot ablating the ability of the fusion protein to promote antibodydependent cell-mediated cytotoxicity (ADCC) or to fix complement. Inparticular, the Fc receptors (FcR) which mediate ADCC (e.g., FcRIII,CD16) exhibit low affinity for immunoglobulin Fc domains, suggestingthat functional binding of Fc to FcR requires avidity stabilization ofthe Fc-FcR complex by virtue of the dimeric structure of heavy chains ina conventional antibody, and/or FcR aggregation and cross-linking by aconventional Ab Fc structure. (Sonderman et al., 2000 Nature 406:267;Radaev et al., 2001 J. Biol. Chem. 276:16469; Radaev et al., 2001 J.Biol. Chem. 276:16478; Koolwijk et al., 1989 J. Immunol. 143:1656; Katoet al., 2000 Immunol. Today 21:310.) Hence, the bindingdomain-immunoglobulin fusion proteins of the present invention providethe advantages associated with single-chain immunoglobulin fusionproteins while also unexpectedly retaining immunological activity.Similarly, the ability to fix complement is typically associated withimmunoglobulins that are dimeric with respect to heavy chain constantregions such as those that comprise Fc, while the bindingdomain-immunoglobulin fusion proteins of the present invention exhibitthe unexpected ability to fix complement.

As noted above, binding domain-immunoglobulin fusion proteins arebelieved, according to non-limiting theory, to be compromised in theirability to dimerize, and further according to theory, this property is aconsequence of a reduction in the number of cysteine residues that arepresent in the immunoglobulin hinge region polypeptide selected forinclusion in the construction of the fusion protein. Determination ofthe relative ability of a polypeptide to dimerize is well within theknowledge of the relevant art, where any of a number of establishedmethodologies may be applied to detect protein dimerization (see, e.g.,Scopes, Protein Purification: Principles and Practice, 1987Springer-Verlag, New York). For example, biochemical separationtechniques for resolving proteins on the basis of molecular size (e.g.,gel electrophoresis, gel filtration chromatography, analyticalultracentrifugation, etc.), and/or comparison of protein physicochemicalproperties before and after introduction of sulfhydryl-active (e.g.,iodoacetamide, N-ethylmaleimide) or disulfide-reducing (e.g.,2-mercaptoethanol, dithiothreitol) agents, or other equivalentmethodologies, may all be employed for determining a degree ofpolypeptide dimerization or oligomerization, and for determiningpossible contribution of disulfide bonds to such potential quarternarystructure. In certain embodiments, the invention relates to a bindingdomain-immunoglobulin fusion protein that exhibits a reduced (i.e., in astatistically significant manner relative to an appropriate IgG-derivedcontrol) ability to dimerize, relative to a wild-type humanimmunoglobulin G hinge region polypeptide as provided herein.Accordingly, those familiar with the art will be able readily todetermine whether a particular fusion protein displays such reducedability to dimerize.

Compositions and methods for preparation of immunoglobulin fusionproteins are well known in the art, as described for example, in U.S.Pat. No. 5,892,019, which discloses recombinant antibodies that are theproducts of a single encoding polynucleotide but which are not bindingdomain-immunoglobulin fusion proteins according to the presentinvention.

For an immunoglobulin fusion protein of the invention which is intendedfor use in humans, the constant regions will typically be of humansequence origin, to minimize a potential anti-human immune response andto provide appropriate effector functions. Manipulation of sequencesencoding antibody constant regions is described in the PCT publicationof Morrison and Oi, WO 89/07142. In particularly preferred embodiments,the CH1 domain is deleted and the carboxyl end of the binding domain, orwhere the binding domain comprises two immunoglobulin variable regionpolypeptides, the second (i.e., more proximal to the C-terminus)variable region is joined to the amino terminus of CH2 through the hingeregion. A schematic diagram depicting the structures of two exemplarybinding domain-immunoglobulin fusion proteins is shown in FIG. 11, whereit should be noted that in particularly preferred embodiments nointerchain disulfide bonds are present, and in other embodiments arestricted number of interchain disulfide bonds may be present relativeto the number of such bonds that would be present if wild-type hingeregion polypeptides were instead present, and that in other embodimentsthe fusion protein comprises a mutated hinge region polypeptide thatexhibits a reduced ability to dimerize, relative to a wild-type humanIgG hinge region polypeptide. Thus, the isolated polynucleotide moleculecodes for a single chain immunoglobulin fusion protein having a bindingdomain that provides specific binding affinity for a selected antigen.

As noted above, in certain embodiments the bindingprotein-immunoglobulin fusion protein comprises at least oneimmunoglobulin variable region polypeptide, which may be a light chainor a heavy chain variable region polypeptide, and in certain embodimentsthe fusion protein comprises at least one such light chain V-region andone such heavy chain V-region and at least one linker peptide that isfused to each of the V-regions. Construction of such binding domains,for example single chain Fv domains, is well known in the art and isdescribed in greater detail in the Examples below, and has beendescribed, for example, in U.S. Pat. No. 5,892,019 and references citedtherein; selection and assembly of single-chain variable regions and oflinker polypeptides that may be fused to each of a heavy chain-derivedand a light chain-derived V region (e.g., to generate a binding domainthat comprises a single-chain Fv polypeptide) is also known to the artand described herein and, for example, in U.S. Pat. No. 5,869,620, U.S.Pat. No. 4,704,692 and U.S. Pat. No. 4,946,778. In certain embodimentsall or a portion of an immunoglobulin sequence that is derived from anon-human source may be “humanized” according to recognized proceduresfor generating humanized antibodies, i.e., immunoglobulin sequences intowhich human Ig sequences are introduced to reduce the degree to which ahuman immune system would perceive such proteins as foreign (see, e.g.,U.S. Pat. Nos. 5,693,762; 5,585,089; 4,816,567; 5,225,539; 5,530,101;and references cited therein).

Once a binding domain-immunoglobulin fusion protein as provided hereinhas been designed, DNAs encoding the polypeptide may be synthesized viaoligonucleotide synthesis as described, for example, in Sinha et al.,Nucleic Acids Res., 12, 4539-4557 (1984); assembled via PCR asdescribed, for example in Innis, Ed., PCR Protocols, Academic Press(1990) and also in Better et al. J. Biol. Chem. 267, 16712-16118 (1992);cloned and expressed via standard procedures as described, for example,in Ausubel et al., Eds., Current Protocols in Molecular Biology, JohnWiley & Sons, New York (1989) and also in Robinson et al., Hum. Antibod.Hybridomas, 2, 84-93 (1991); and tested for specific antigen bindingactivity, as described, for example, in Harlow et al., Eds., Antibodies:A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, ColdSpring Harbor (1988) and Munson et al., Anal. Biochem., 107, 220-239(1980).

The preparation of single polypeptide chain binding molecules of the Fvregion, single-chain Fv molecules, is described in U.S. Pat. No.4,946,778, which is incorporated herein by reference. In the presentinvention, single-chain Fv-like molecules are synthesized by encoding afirst variable region of the heavy or light chain, followed by one ormore linkers to the variable region of the corresponding light or heavychain, respectively. The selection of appropriate linker(s) between thetwo variable regions is described in U.S. Pat. No. 4,946,778. Anexemplary linker described herein is (Gly-Gly-Gly-Gly-Ser)₃. The linkeris used to convert the naturally aggregated but chemically separateheavy and light chains into the amino terminal antigen binding portionof a single polypeptide chain, wherein this antigen binding portion willfold into a structure similar to the original structure made of twopolypeptide chains and thus retain the ability to bind to the antigen ofinterest. The nucleotide sequences encoding the variable regions of theheavy and light chains, joined by a sequence encoding a linker, arejoined to a nucleotide sequence encoding antibody constant regions. Theconstant regions are those which permit the resulting polypeptide toform interchain disulfide bonds to form a dimer, and which containdesired effector functions, such as the ability to mediateantibody-dependent cellular cytotoxicity (ADCC). For animmunoglobulin-like molecule of the invention which is intended for usein humans, the constant regions will typically be substantially human tominimize a potential anti-human immune response and to provideappropriate effector functions. Manipulation of sequences encodingantibody constant regions is described in the PCT publication ofMorrison and Oi, WO 89/07142, which is incorporated herein by reference.In preferred embodiments, the CH1 domain is deleted and the carboxyl endof the second variable region is joined to the amino terminus of CH2through the hinge region. The Cys residue of the hinge which makes adisulfide bond with a corresponding Cys of the light chain, to hold theheavy and light chains of the native antibody molecule, can be deletedor, preferably, is substituted with, e.g., a Pro residue or the like.

As described above, the present invention provides recombinantexpression constructs capable of directing the expression of bindingdomain-immunoglobulin fusion proteins as provided herein. The aminoacids, which occur in the various amino acid sequences referred toherein, are identified according to their well known three letter or oneletter abbreviations. The nucleotides, which occur in the various DNAsequences or fragments thereof referred herein, are designated with thestandard single letter designations used routinely in the art. A givenamino acid sequence may also encompass similar amino acid sequenceshaving only minor changes, for example by way of illustration and notlimitation, covalent chemical modifications, insertions, deletions andsubstitutions, which may further include conservative substitutions.Amino acid sequences that are similar to one another may sharesubstantial regions of sequence homology. In like fashion, nucleotidesequences may encompass substantially similar nucleotide sequenceshaving only minor changes, for example by way of illustration and notlimitation, covalent chemical modifications, insertions, deletions andsubstitutions, which may further include silent mutations owing todegeneracy of the genetic code. Nucleotide sequences that are similar toone another may share substantial regions of sequence homology.

The presence of a malignant condition in a subject refers to thepresence of dysplastic, cancerous and/or transformed cells in thesubject, including, for example neoplastic, tumor, non-contact inhibitedor oncogenically transformed cells, or the like. In preferredembodiments contemplated by the present invention, for example, suchcancer cells are malignant hematopoietic cells, such as transformedcells of lymphoid lineage and in particular, B-cell lymphomas and thelike; cancer cells may in certain preferred embodiments also beepithelial cells such as carcinoma cells. The invention alsocontemplates B-cell disorders, which may include certain malignantconditions that affect B-cells (e.g., B-cell lymphoma) but which is notintended to be so limited, and which is also intended to encompassautoimmune diseases and in particular, diseases, disorders andconditions that are characterized by autoantibody production.

Autoantibodies are antibodies that react with self antigens.Autoantibodies are detected in several autoimmune diseases (i.e., adisease, disorder or condition wherein a host immune system generates aninappropriate anti-“self” immune reaction) where they are involved indisease activity. The current treatments for these autoimmune diseasesare immunosuppressive drugs that require continuing administration, lackspecificity, and cause significant side effects. New approaches that caneliminate autoantibody production with minimal toxicity will address anunmet medical need for a spectrum of diseases that affect many people.The subject invention binding domain-immunoglobulin fusion protein isdesigned for improved penetration into lymphoid tissues. Depletion of Blymphocytes interrupts the autoantibody production cycle, and allows theimmune system to reset as new B lymphocytes are produced from precursorsin the bone marrow.

A number of diseases have been identified for which beneficial effectsare believed, according to non-limiting theory, to result from B celldepletion therapy; a brief description of several exemplars of thesediseases follows.

Autoimmune thyroid disease includes Graves' disease and Hashimoto'sthyroiditis. In the United States alone, there are about 20 millionpeople who have some form of autoimmune thyroid disease. Autoimmunethyroid disease results from the production of autoantibodies thateither stimulate the thyroid to cause hyperthyroidism (Graves' disease)or destroy the thyroid to cause hypothyroidism (Hashimoto'sthyroiditis). Stimulation of the thyroid is caused by autoantibodiesthat bind and activate the thyroid stimulating hormone (TSH) receptor.Destruction of the thyroid is caused by autoantibodies that react withother thyroid antigens.

Current therapy for Graves' disease includes surgery, radioactiveiodine, or antithyroid drug therapy. Radioactive iodine is widely used,since antithyroid medications have significant side effects and diseaserecurrence is high. Surgery is reserved for patients with large goitersor where there is a need for very rapid normalization of thyroidfunction. There are no therapies that target the production ofautoantibodies responsible for stimulating the TSH receptor. Currenttherapy for Hashimoto's thyroiditis is levothyroxine sodium, and therapyis usually lifelong because of the low likelihood of remission.Suppressive therapy has been shown to shrink goiters in Hashimoto'sthryoiditis, but no therapies that reduce autoantibody production totarget the disease mechanism are known.

Rheumatoid arthritis (RA) is a chronic disease characterized byinflammation of the joints, leading to swelling, pain, and loss offunction. RA effects an estimated 2.5 million people in the UnitedStates. RA is caused by a combination of events including an initialinfection or injury, an abnormal immune response, and genetic factors.While autoreactive T cells and B cells are present in RA, the detectionof high levels of antibodies that collect in the joints, calledrheumatoid factor, is used in the diagnosis of RA. Current therapy forRA includes many medications for managing pain and slowing theprogression of the disease. No therapy has been found that can cure thedisease. Medications include nonsteroidal antiinflammatory drugs(NSAIDS), and disease modifying antirheumatic drugs (DMARDS). NSAIDS areeffective in benign disease, but fail to prevent the progression tojoint destruction and debility in severe RA. Both NSAIDS and DMARDS areassociated with signficant side effects. Only one new DMARD,Leflunomide, has been approved in over 10 years. Leflunomide blocksproduction of autoantibodies, reduces inflammation, and slowsprogression of RA. However, this drug also causes severe side effectsincluding nausea, diarrhea, hair loss, rash, and liver injury.

Systemic Lupus Erythematosus (SLE) is an autoimmune disease caused byrecurrent injuries to blood vessels in multiple organs, including thekidney, skin, and joints. SLE effects over 500,000 people in the UnitedStates. In patients with SLE, a faulty interaction between T cells and Bcells results in the production of autoantibodies that attack the cellnucleus. These include anti-double stranded DNA and anti-Sm antibodies.Autoantibodies that bind phospholipids are also found in about half ofSLE patients, and are responsible for blood vessel damage and low bloodcounts. Immune complexes accumulate the kidneys, blood vessels, andjoints of SLE patients, where they cause inflammation and tissue damage.No treatment for SLE has been found to cure the disease. NSAIDS andDMARDS are used for therapy depending upon the severity of the disease.Plasmapheresis with plasma exchange to remove autoantibodies can causetemporary improvement in SLE patients. There is general agreement thatautoantibodies are responsible for SLE, so new therapies that depletethe B cell lineage, allowing the immune system to reset as new B cellsare generated from precursors, offer hope for long lasting benefit inSLE patients.

Sjogrens syndrome is an autoimmune disease characterized by destructionof the body's moisture producing glands. Sjogrens syndrome is one of themost prevalent autoimmune disorders, striking up to 4 million people inthe United States. About half of people with Sjogren's also have aconnective tissue disease, such as rheumatoid arthritis, while the otherhalf have primary Sjogren's with no other concurrent autoimmune disease.Autoantibodies, including anti-nuclear antibodies, rheumatoid factor,anti-fodrin, and anti-muscarinic receptor are often present in patientswith Sjogrens syndrome. Conventional therapy includes corticosteroids.

Immune Thrombocytopenic purpura (ITP) is caused by autoantibodies thatbind to blood platelets and cause their destruction. Some cases of ITPare caused by drugs, and others are associated with infection,pregnancy, or autoimmune disease such as SLE. About half of all casesare classified as “idiopathic”, meaning the cause is unknown. Thetreatment of ITP is determined by the severity of the symptoms. In somecases, no therapy is needed. In most cases, immunosuppressive drugs,including corticosteroids or intravenous infusions of immune globulin todeplete T cells. Another treatment that usually results in an increasednumber of platelets is removal of the spleen, the organ that destroysantibody-coated platelets. More potent immunosuppressive drugs,including cyclosporine, cyclophosphamide, or azathioprine are used forpatients with severe cases. Removal of autoantibodies by passage ofpatients' plasma over a Protein A column is used as a second linetreatment in patients with severe disease.

Multiple Sclerosis (MS) is an autoimmune disease characterized byinflammation of the central nervous system and destruction of myelin,which insulates nerve cell fibers in the brain, spinal cord, and body.Although the cause of MS is unknown, it is widely believed thatautoimmune T cells are primary contributors to the pathogenesis of thedisease. However, high levels of antibodies are present in the cerebralspinal fluid of patients with MS, and some theories predict that the Bcell response leading to antibody production is important for mediatingthe disease. No B cell depletion therapies have been studies in patientswith MS. There is no cure for MS. Current therapy is corticosteroids,which can reduce the duration and severity of attacks, but do not affectthe course of MS over time. New biotechnology interferon (IFN) therapiesfor MS have recently been approved.

Myasthenia Gravis (MG) is a chronic autoimmune neuromuscular disorderthat is characterized by weakness of the voluntary muscle groups. MGeffects about 40,000 people in the United States. MG is caused byautoantibodies that bind to acetylcholine receptors expressed atneuromuscular junctions. The autoantibodies reduce or blockacetylcholine receptors, preventing the transmission of signals fromnerves to muscles. There is no known cure for MG. Common treatmentsinclude immunosuppression with corticosteroids, cyclosporine,cyclophosphamide, or azathioprine. Surgical removal of the thymus isoften used to blunt the autoimmune response. Plasmapheresis, used toreduce autoantibody levels in the blood, is effective in MG, but isshort-lived because the production of autoantibodies continues.Plasmapheresis is usually reserved for severe muscle weakness prior tosurgery.

Psoriasis effects approximately five million people. Autoimmuneinflammation in the skin. Psoriasis associated with arthritis in 30%(psoriatic arthritis). Many treatments, including steroids, UV lightretenoids, vitamin D derivatives, cyclosporine, methotrexate.

Scleroderma is a chronic autoimmune disease of the connective tissuethat is also known as systemic sclerosis. Scleroderma is characterizedby an overproduction of collagen, resulting in a thickening of the skin.Approximately 300,000 people in the United States have scleroderma.

Inflammatory Bowel Disease including Crohn's disease and Ulcerativecolitis, are autoimmune diseases of the digestive system.

The present invention further relates to constructs encoding bindingdomain-immunoglobulin fusion proteins, and in particular to methods foradministering recombinant constructs encoding such proteins that may beexpressed, for example, as fragments, analogs and derivatives of suchpolypeptides. The terms “fragment,” “derivative” and “analog” whenreferring to binding domain-immunoglobulin fusion polypeptides or fusionproteins, refers to any binding domain-immunoglobulin fusion polypeptideor fusion protein that retains essentially the same biological functionor activity as such polypeptide. Thus, an analog includes a proproteinwhich can be activated by cleavage of the proprotein portion to producean active binding domain-immunoglobulin fusion polypeptide.

A fragment, derivative or analog of an binding domain-immunoglobulinfusion polypeptide or fusion protein, including bindingdomain-immunoglobulin fusion polypeptides or fusion proteins encoded bythe cDNAs referred to herein, may be (i) one in which one or more of theamino acid residues are substituted with a conserved or non-conservedamino acid residue (preferably a conserved amino acid residue) and suchsubstituted amino acid residue may or may not be one encoded by thegenetic code, or (ii) one in which one or more of the amino acidresidues includes a substituent group, or (iii) one in which additionalamino acids are fused to the binding domain-immunoglobulin fusionpolypeptide, including amino acids that are employed for detection orspecific functional alteration of the binding domain-immunoglobulinfusion polypeptide or a proprotein sequence. Such fragments, derivativesand analogs are deemed to be within the scope of those skilled in theart from the teachings herein.

The polypeptides of the present invention include bindingdomain-immunoglobulin fusion polypeptides and fusion proteins havingbinding domain polypeptide amino acid sequences that are identical orsimilar to sequences known in the art, or fragments or portions thereof.For example by way of illustration and not limitation, the human CD154molecule extracellular domain is contemplated for use according to theinstant invention, as are polypeptides having at least 70% similarity(preferably a 70% identity) and more preferably 90% similarity (morepreferably a 90% identity) to the reported polypeptide and still morepreferably a 95% similarity (still more preferably a 95% identity) tothe reported polypeptides and to portions of such polypeptides, whereinsuch portions of a binding domain-immunoglobulin fusion polypeptidegenerally contain at least 30 amino acids and more preferably at least50 amino acids.

As known in the art “similarity” between two polypeptides is determinedby comparing the amino acid sequence and conserved amino acidsubstitutes thereto of the polypeptide to the sequence of a secondpolypeptide. Fragments or portions of the nucleic acids encodingpolypeptides of the present invention may be used to synthesizefull-length nucleic acids of the present invention. As used herein, “%identity” refers to the percentage of identical amino acids situated atcorresponding amino acid residue positions when two or more polypeptideare aligned and their sequences analyzed using a gapped BLAST algorithm(e.g., Altschul et al., 1997 Nucl. Ac. Res. 25:3389) which weightssequence gaps and sequence mismatches according to the defaultweightings provided by the National Institutes of Health/NCBI database(National Center for Biotechnology Information, National Library ofMedicine, Building 38A, Bethesda, Md. 20894).

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally occurring nucleic acid orpolypeptide present in a living animal is not isolated, but the samenucleic acid or polypeptide, separated from some or all of theco-existing materials in the natural system, is isolated. Such nucleicacids could be part of a vector and/or such nucleic acids orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region “leader and trailer” as well as intervening sequences(introns) between individual coding segments (exons).

As described herein, the invention provides bindingdomain-immunoglobulin fusion proteins encoded by nucleic acids that havethe binding domain coding sequence fused in frame to an additionalimmunoglobulin domain encoding sequence to provide for expression of abinding domain polypeptide sequence fused to an additional functionalpolypeptide sequence that permits, for example by way of illustrationand not limitation, detection, functional alteration, isolation and/orpurification of the fusion protein. Such fusion proteins may permitfunctional alteration of a binding domain by containing additionalimmunoglobulin-derived polypeptide sequences that influence behavior ofthe fusion product, for example (and as described above) by reducing theavailability of sufhydryl groups for participation in disulfide bondformation, and by conferring the ability to potentiate ADCC and/or CDC.

Modification of the polypeptide may be effected by any means known tothose of skill in this art. The preferred methods herein rely onmodification of DNA encoding the fusion protein and expression of themodified DNA. DNA encoding one of the binding domain-immunoglobulinfusions discussed above may be mutagenized using standard methodologies,including those described below. For example, cysteine residues that mayotherwise facilitate multimer formation or promote particular molecularconformations can be deleted from a polypeptide or replaced, e.g.,cysteine residues that are responsible for aggregate formation. Ifnecessary, the identity of cysteine residues that contribute toaggregate formation may be determined empirically, by deleting and/orreplacing a cysteine residue and ascertaining whether the resultingprotein aggregates in solutions containing physiologically acceptablebuffers and salts. In addition, fragments of bindingdomain-immunoglobulin fusions may be constructed and used. As notedabove, the counterreceptor/ligand binding domains for many candidatebinding domain-immunoglobulin fusion have been delineated, such that onehaving ordinary skill in the art may readily select appropriatepolypeptide domains for inclusion in the encoded products of the instantexpression constructs.

Conservative substitutions of amino acids are well-known and may be madegenerally without altering the biological activity of the resultingbinding domain-immunoglobulin fusion protein molecule. For example, suchsubstitutions are generally made by interchanging within the groups ofpolar residues, charged residues, hydrophobic residues, small residues,and the like. If necessary, such substitutions may be determinedempirically merely by testing the resulting modified protein for theability to bind to the appropriate cell surface receptors in in vitrobiological assays, or to bind to appropriate antigens or desired targetmolecules.

The present invention further relates to nucleic acids which hybridizeto binding domain-immunoglobulin fusion protein encoding polynucleotidesequences as provided herein, or their complements, as will be readilyapparent to those familiar with the art, if there is at least 70%,preferably at least 90%, and more preferably at least 95% identitybetween the sequences. The present invention particularly relates tonucleic acids which hybridize under stringent conditions to the bindingdomain-immunoglobulin fusion encoding nucleic acids referred to herein.As used herein, the term “stringent conditions” means hybridization willoccur only if there is at least 95% and preferably at least 97% identitybetween the sequences. The nucleic acids which hybridize to bindingdomain-immunoglobulin fusion encoding nucleic acids referred to herein,in preferred embodiments, encode polypeptides which retain substantiallythe same biological function or activity as the bindingdomain-immunoglobulin fusion polypeptides encoded by the cDNAs of thereferences cited herein.

As used herein, to “hybridize” under conditions of a specifiedstringency is used to describe the stability of hybrids formed betweentwo single-stranded nucleic acid molecules. Stringency of hybridizationis typically expressed in conditions of ionic strength and temperatureat which such hybrids are annealed and washed. Typically “high”,“medium” and “low” stringency encompass the following conditions orequivalent conditions thereto: high stringency: 0.1×SSPE or SSC, 0.1%SDS, 65° C.; medium stringency: 0.2×SSPE or SSC, 0.1% SDS, 50° C.; andlow stringency: 1.0×SSPE or SSC, 0.1% SDS, 50° C. As known to thosehaving ordinary skill in the art, variations in stringency ofhybridization conditions may be achieved by altering the time,temperature and/or concentration of the solutions used forprehybridization, hybridization and wash steps, and suitable conditionsmay also depend in part on the particular nucleotide sequences of theprobe used, and of the blotted, proband nucleic acid sample.Accordingly, it will be appreciated that suitably stringent conditionscan be readily selected without undue experimentation where a desiredselectivity of the probe is identified, based on its ability tohybridize to one or more certain proband sequences while not hybridizingto certain other proband sequences.

The nucleic acids of the present invention, also referred to herein aspolynucleotides, may be in the form of RNA or in the form of DNA, whichDNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may bedouble-stranded or single-stranded, and if single stranded may be thecoding strand or non-coding (anti-sense) strand. A coding sequence whichencodes an binding domain-immunoglobulin fusion polypeptide for useaccording to the invention may be identical to the coding sequence knownin the art for any given binding domain-immunoglobulin fusion, or may bea different coding sequence, which, as a result of the redundancy ordegeneracy of the genetic code, encodes the same bindingdomain-immunoglobulin fusion polypeptide.

The nucleic acids which encode binding domain-immunoglobulin fusionpolypeptides for use according to the invention may include, but are notlimited to: only the coding sequence for the bindingdomain-immunoglobulin fusion polypeptide; the coding sequence for thebinding domain-immunoglobulin fusion polypeptide and additional codingsequence; the coding sequence for the binding domain-immunoglobulinfusion polypeptide (and optionally additional coding sequence) andnon-coding sequence, such as introns or non-coding sequences 5′ and/or3′ of the coding sequence for the binding domain-immunoglobulin fusionpolypeptide, which for example may further include but need not belimited to one or more regulatory nucleic acid sequences that may be aregulated or regulatable promoter, enhancer, other transcriptionregulatory sequence, repressor binding sequence, translation regulatorysequence or any other regulatory nucleic acid sequence. Thus, the term“nucleic acid encoding” or “polynucleotide encoding” a bindingdomain-immunoglobulin fusion protein encompasses a nucleic acid whichincludes only coding sequence for a binding domain-immunoglobulin fusionpolypeptide as well as a nucleic acid which includes additional codingand/or non-coding sequence(s).

Nucleic acids and oligonucleotides for use as described herein can besynthesized by any method known to those of skill in this art (see,e.g., WO 93/01286, U.S. application Ser. No. 07/723,454; U.S. Pat. No.5,218,088; U.S. Pat. No. 5,175,269; U.S. Pat. No. 5,109,124).Identification of oligonucleotides and nucleic acid sequences for use inthe present invention involves methods well known in the art. Forexample, the desirable properties, lengths and other characteristics ofuseful oligonucleotides are well known. In certain embodiments,synthetic oligonucleotides and nucleic acid sequences may be designedthat resist degradation by endogenous host cell nucleolytic enzymes bycontaining such linkages as: phosphorothioate, methylphosphonate,sulfone, sulfate, ketyl, phosphorodithioate, phosphoramidate, phosphateesters, and other such linkages that have proven useful in antisenseapplications (see, e.g., Agrwal et al., Tetrehedron Lett. 28:3539-3542(1987); Miller et al., J. Am. Chem. Soc. 93:6657-6665 (1971); Stec etal., Tetrehedron Lett. 26:2191-2194 (1985); Moody et al., Nucl. AcidsRes. 12:4769-4782 (1989); Uznanski et al., Nucl. Acids Res. (1989);Letsinger et al., Tetrahedron 40:137-143 (1984); Eckstein, Annu. Rev.Biochem. 54:367-402 (1985); Eckstein, Trends Biol. Sci. 14:97-100(1989); Stein In: Oligodeoxynucleotides. Antisense Inhibitors of GeneExpression, Cohen, Ed, Macmillan Press, London, pp. 97-117 (1989); Jageret al., Biochemistry 27:7237-7246 (1988)).

In one embodiment, the present invention provides truncated components(e.g., binding domain polypeptide, hinge region polypeptide, linker,etc.) for use in a binding domain-immunoglobulin fusion protein, and inanother embodiment the invention provides nucleic acids encoding abinding domain-immunoglobulin fusion protein having such truncatedcomponents. A truncated molecule may be any molecule that comprises lessthan a full length version of the molecule. Truncated molecules providedby the present invention may include truncated biological polymers, andin preferred embodiments of the invention such truncated molecules maybe truncated nucleic acid molecules or truncated polypeptides. Truncatednucleic acid molecules have less than the full length nucleotidesequence of a known or described nucleic acid molecule, where such aknown or described nucleic acid molecule may be a naturally occurring, asynthetic or a recombinant nucleic acid molecule, so long as one skilledin the art would regard it as a full length molecule. Thus, for example,truncated nucleic acid molecules that correspond to a gene sequencecontain less than the full length gene where the gene comprises codingand non-coding sequences, promoters, enhancers and other regulatorysequences, flanking sequences and the like, and other functional andnon-functional sequences that are recognized as part of the gene. Inanother example, truncated nucleic acid molecules that correspond to amRNA sequence contain less than the full length mRNA transcript, whichmay include various translated and non-translated regions as well asother functional and non-functional sequences.

In other preferred embodiments, truncated molecules are polypeptidesthat comprise less than the full length amino acid sequence of aparticular protein or polypeptide component. As used herein “deletion”has its common meaning as understood by those familiar with the art, andmay refer to molecules that lack one or more of a portion of a sequencefrom either terminus or from a non-terminal region, relative to acorresponding full length molecule, for example, as in the case oftruncated molecules provided herein. Truncated molecules that are linearbiological polymers such as nucleic acid molecules or polypeptides mayhave one or more of a deletion from either terminus of the molecule or adeletion from a non-terminal region of the molecule, where suchdeletions may be deletions of 1-1500 contiguous nucleotide or amino acidresidues, preferably 1-500 contiguous nucleotide or amino acid residuesand more preferably 1-300 contiguous nucleotide or amino acid residues.In certain particularly preferred embodiments truncated nucleic acidmolecules may have a deletion of 270-330 contiguous nucleotides. Incertain other particularly preferred embodiments truncated polypeptidemolecules may have a deletion of 80-140 contiguous amino acids.

The present invention further relates to variants of the hereinreferenced nucleic acids which encode fragments, analogs and/orderivatives of a binding domain-immunoglobulin fusion polypeptide. Thevariants of the nucleic acids encoding binding domain-immunoglobulinfusion may be naturally occurring allelic variants of the nucleic acidsor non-naturally occurring variants. As is known in the art, an allelicvariant is an alternate form of a nucleic acid sequence which may haveat least one of a substitution, a deletion or an addition of one or morenucleotides, any of which does not substantially alter the function ofthe encoded binding domain-immunoglobulin fusion polypeptide.

Variants and derivatives of binding domain-immunoglobulin fusion may beobtained by mutations of nucleotide sequences encoding bindingdomain-immunoglobulin fusion polypeptides. Alterations of the nativeamino acid sequence may be accomplished by any of a number ofconventional methods. Mutations can be introduced at particular loci bysynthesizing oligonucleotides containing a mutant sequence, flanked byrestriction sites enabling ligation to fragments of the native sequence.Following ligation, the resulting reconstructed sequence encodes ananalog having the desired amino acid insertion, substitution, ordeletion.

Alternatively, oligonucleotide-directed site-specific mutagenesisprocedures can be employed to provide an altered gene whereinpredetermined codons can be altered by substitution, deletion orinsertion. Exemplary methods of making such alterations are disclosed byWalder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985);Craik (BioTechniques, January 1985, 12-19); Smith et al. (GeneticEngineering: Principles and Methods BioTechniques, January 1985, 12-19);Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press,1981); Kunkel (Proc. Natl. Acad. Sci. USA 82:488, 1985); Kunkel et al.(Methods in Enzymol. 154:367, 1987); and U.S. Pat. Nos. 4,518,584 and4,737,462.

As an example, modification of DNA may be performed by site-directedmutagenesis of DNA encoding the protein combined with the use of DNAamplification methods using primers to introduce and amplify alterationsin the DNA template, such as PCR splicing by overlap extension (SOE).Site-directed mutagenesis is typically effected using a phage vectorthat has single- and double-stranded forms, such as M13 phage vectors,which are well-known and commercially available. Other suitable vectorsthat contain a single-stranded phage origin of replication may be used(see, e.g., Veira et al., Meth. Enzymol. 15:3, 1987). In general,site-directed mutagenesis is performed by preparing a single-strandedvector that encodes the protein of interest (e.g., all or a componentportion of a given binding domain-immunoglobulin fusion protein). Anoligonucleotide primer that contains the desired mutation within aregion of homology to the DNA in the single-stranded vector is annealedto the vector followed by addition of a DNA polymerase, such as E. coliDNA polymerase I (Klenow fragment), which uses the double strandedregion as a primer to produce a heteroduplex in which one strand encodesthe altered sequence and the other the original sequence. Theheteroduplex is introduced into appropriate bacterial cells and clonesthat include the desired mutation are selected. The resulting alteredDNA molecules may be expressed recombinantly in appropriate host cellsto produce the modified protein.

Equivalent DNA constructs that encode various additions or substitutionsof amino acid residues or sequences, or deletions of terminal orinternal residues or sequences not needed for biological activity arealso encompassed by the invention. For example, and as discussed above,sequences encoding Cys residues that are not desirable or essential forbiological activity can be altered to cause the Cys residues to bedeleted or replaced with other amino acids, preventing formation ofincorrect intramolecular disulfide bridges upon renaturation.

Host organisms include those organisms in which recombinant productionof binding domain-immunoglobulin fusion products encoded by therecombinant constructs of the present invention may occur, such asbacteria (for example, E. coli), yeast (for example, Saccharomycescerevisiae and Pichia pastoris), insect cells and mammals, including invitro and in vivo expression. Host organisms thus may include organismsfor the construction, propagation, expression or other steps in theproduction of the compositions provided herein; hosts also includesubjects in which immune responses take place, as described above.Presently preferred host organisms are E. coli bacterial strains, inbredmurine strains and murine cell lines, and human cells, subjects and celllines.

The DNA construct encoding the desired binding domain-immunoglobulinfusion is introduced into a plasmid for expression in an appropriatehost. In preferred embodiments, the host is a bacterial host. Thesequence encoding the ligand or nucleic acid binding domain ispreferably codon-optimized for expression in the particular host. Thus,for example, if a human binding domain-immunoglobulin fusion isexpressed in bacteria, the codons would be optimized for bacterialusage. For small coding regions, the gene can be synthesized as a singleoligonucleotide. For larger proteins, splicing of multipleoligonucleotides, mutagenesis, or other techniques known to those in theart may be used. The sequences of nucleotides in the plasmids that areregulatory regions, such as promoters and operators, are operationallyassociated with one another for transcription. The sequence ofnucleotides encoding a binding domain-immunoglobulin fusion protein mayalso include DNA encoding a secretion signal, whereby the resultingpeptide is a precursor protein. The resulting processed protein may berecovered from the periplasmic space or the fermentation medium.

In preferred embodiments, the DNA plasmids also include a transcriptionterminator sequence. As used herein, a “transcription terminator region”is a sequence that signals transcription termination. The entiretranscription terminator may be obtained from a protein-encoding gene,which may be the same or different from the inserted bindingdomain-immunoglobulin fusion encoding gene or the source of thepromoter. Transcription terminators are optional components of theexpression systems herein, but are employed in preferred embodiments.

The plasmids used herein include a promoter in operative associationwith the DNA encoding the protein or polypeptide of interest and aredesigned for expression of proteins in a suitable host as describedabove (e.g., bacterial, murine or human) depending upon the desired useof the plasmid (e.g., administration of a vaccine containing bindingdomain-immunoglobulin fusion encoding sequences). Suitable promoters forexpression of proteins and polypeptides herein are widely available andare well known in the art. Inducible promoters or constitutive promotersthat are linked to regulatory regions are preferred. Such promotersinclude, but are not limited to, the T7 phage promoter and other T7-likephage promoters, such as the T3, T5 and SP6 promoters, the trp, lpp, andlac promoters, such as the lacUV5, from E. coli; the P10 or polyhedringene promoter of baculovirus/insect cell expression systems (see, e.g.,U.S. Pat. Nos. 5,243,041, 5,242,687, 5,266,317, 4,745,051, and5,169,784) and inducible promoters from other eukaryotic expressionsystems. For expression of the proteins such promoters are inserted in aplasmid in operative linkage with a control region such as the lacoperon.

Preferred promoter regions are those that are inducible and functionalin E. coli. Examples of suitable inducible promoters and promoterregions include, but are not limited to: the E. coli lac operatorresponsive to isopropyl β-D-thiogalactopyranoside (IPTG; see Nakamura etal., Cell 18:1109-1117, 1979); the metallothionein promotermetal-regulatory-elements responsive to heavy-metal (e.g., zinc)induction (see, e.g., U.S. Pat. No. 4,870,009 to Evans et al.); thephage T7lac promoter responsive to IPTG (see, e.g., U.S. Pat. No.4,952,496; and Studier et al., Meth. Enzymol. 185:60-89, 1990) and theTAC promoter.

The plasmids may optionally include a selectable marker gene or genesthat are functional in the host. A selectable marker gene includes anygene that confers a phenotype on bacteria that allows transformedbacterial cells to be identified and selectively grown from among a vastmajority of untransformed cells. Suitable selectable marker genes forbacterial hosts, for example, include the ampicillin resistance gene(Amp^(r)), tetracycline resistance gene (Tc^(r)) and the kanamycinresistance gene (Kan^(r)). The kanamycin resistance gene is presentlypreferred.

The plasmids may also include DNA encoding a signal for secretion of theoperably linked protein. Secretion signals suitable for use are widelyavailable and are well known in the art. Prokaryotic and eukaryoticsecretion signals functional in E. coli may be employed. The presentlypreferred secretion signals include, but are not limited to, thoseencoded by the following E. coli genes: ompA, ompT, ompF, ompC,beta-lactamase, and alkaline phosphatase, and the like (von Heijne, J.Mol. Biol. 184:99-105, 1985). In addition, the bacterial pelB genesecretion signal (Lei et al., J. Bacteriol. 169:4379, 1987), the phoAsecretion signal, and the cek2 functional in insect cell may beemployed. The most preferred secretion signal is the E. coli ompAsecretion signal. Other prokaryotic and eukaryotic secretion signalsknown to those of skill in the art may also be employed (see, e.g., vonHeijne, J. Mol. Biol. 184:99-105, 1985). Using the methods describedherein, one of skill in the art can substitute secretion signals thatare functional in either yeast, insect or mammalian cells to secreteproteins from those cells.

Preferred plasmids for transformation of E. coli cells include the pETexpression vectors (e.g., pET-11a, pET-12a-c, pET-15b; see U.S. Pat. No.4,952,496; available from Novagen, Madison, Wis.). Other preferredplasmids include the pKK plasmids, particularly pKK 223-3, whichcontains the tac promoter (Brosius et al., Proc. Natl. Acad. Sci.81:6929, 1984; Ausubel et al., Current Protocols in Molecular Biology;U.S. Pat. Nos. 5,122,463, 5,173,403, 5,187,153, 5,204,254, 5,212,058,5,212,286, 5,215,907, 5,220,013, 5,223,483, and 5,229,279). Plasmid pKKhas been modified by replacement of the ampicillin resistance gene witha kanamycin resistance gene. (Available from Pharmacia; obtained frompUC4K, see, e.g., Vieira et al. (Gene 19:259-268, 1982; and U.S. Pat.No. 4,719,179.) Baculovirus vectors, such as pBlueBac (also calledpJVETL and derivatives thereof), particularly pBlueBac III (see, e.g.,U.S. Pat. Nos. 5,278,050, 5,244,805, 5,243,041, 5,242,687, 5,266,317,4,745,051, and 5,169,784; available from Invitrogen, San Diego) may alsobe used for expression of the polypeptides in insect cells. Otherplasmids include the pIN-IIIompA plasmids (see U.S. Pat. No. 4,575,013;see also Duffaud et al., Meth. Enz. 153:492-507, 1987), such aspIN-IIIompA2.

Preferably, the DNA molecule is replicated in bacterial cells,preferably in E. coli. The preferred DNA molecule also includes abacterial origin of replication, to ensure the maintenance of the DNAmolecule from generation to generation of the bacteria. In this way,large quantities of the DNA molecule can be produced by replication inbacteria. Preferred bacterial origins of replication include, but arenot limited to, the f1-ori and col E1 origins of replication. Preferredhosts contain chromosomal copies of DNA encoding T7 RNA polymeraseoperably linked to an inducible promoter, such as the lacUV promoter(see U.S. Pat. No. 4,952,496). Such hosts include, but are not limitedto, lysogens E. coli strains HMS174(DE3)pLysS, BL21(DE3)pLysS,HMS174(DE3) and BL21(DE3). Strain BL21(DE3) is preferred. The pLysstrains provide low levels of T7 lysozyme, a natural inhibitor of T7 RNApolymerase.

The DNA molecules provided may also contain a gene coding for arepressor protein. The repressor protein is capable of repressing thetranscription of a promoter that contains sequences of nucleotides towhich the repressor protein binds. The promoter can be derepressed byaltering the physiological conditions of the cell. For example, thealteration can be accomplished by adding to the growth medium a moleculethat inhibits the ability to interact with the operator or withregulatory proteins or other regions of the DNA or by altering thetemperature of the growth media. Preferred repressor proteins include,but are not limited to the E. coli lacI repressor responsive to IPTGinduction, the temperature sensitive λ cI857 repressor, and the like.The E. coli lacI repressor is preferred.

In general, recombinant constructs of the subject invention will alsocontain elements necessary for transcription and translation. Inparticular, such elements are preferred where the recombinant expressionconstruct containing nucleic acid sequences encoding bindingdomain-immunoglobulin fusion proteins is intended for expression in ahost cell or organism. In certain embodiments of the present invention,cell type preferred or cell type specific expression of a cell bindingdomain-immunoglobulin fusion encoding gene may be achieved by placingthe gene under regulation of a promoter. The choice of the promoter willdepend upon the cell type to be transformed and the degree or type ofcontrol desired. Promoters can be constitutive or active and may furtherbe cell type specific, tissue specific, individual cell specific, eventspecific, temporally specific or inducible. Cell-type specific promotersand event type specific promoters are preferred. Examples ofconstitutive or nonspecific promoters include the SV40 early promoter(U.S. Pat. No. 5,118,627), the SV40 late promoter (U.S. Pat. No.5,118,627), CMV early gene promoter (U.S. Pat. No. 5,168,062), andadenovirus promoter. In addition to viral promoters, cellular promotersare also amenable within the context of this invention. In particular,cellular promoters for the so-called housekeeping genes are useful.Viral promoters are preferred, because generally they are strongerpromoters than cellular promoters. Promoter regions have been identifiedin the genes of many eukaryotes including higher eukaryotes, such thatsuitable promoters for use in a particular host can be readily selectedby those skilled in the art.

Inducible promoters may also be used. These promoters include MMTV LTR(PCT WO 91/13160), inducible by dexamethasone; metallothionein promoter,inducible by heavy metals; and promoters with cAMP response elements,inducible by cAMP. By using an inducible promoter, the nucleic acidsequence encoding a binding domain-immunoglobulin fusion protein may bedelivered to a cell by the subject invention expression construct andwill remain quiescent until the addition of the inducer. This allowsfurther control on the timing of production of the gene product.

Event-type specific promoters are active or up-regulated only upon theoccurrence of an event, such as tumorigenicity or viral infection. TheHIV LTR is a well known example of an event-specific promoter. Thepromoter is inactive unless the tat gene product is present, whichoccurs upon viral infection. Some event-type promoters are alsotissue-specific.

Additionally, promoters that are coordinately regulated with aparticular cellular gene may be used. For example, promoters of genesthat are coordinately expressed may be used when expression of aparticular binding domain-immunoglobulin fusion protein-encoding gene isdesired in concert with expression of one or more additional endogenousor exogenously introduced genes. This type of promoter is especiallyuseful when one knows the pattern of gene expression relevant toinduction of an immune response in a particular tissue of the immunesystem, so that specific immunocompetent cells within that tissue may beactivated or otherwise recruited to participate in the immune response.

In addition to the promoter, repressor sequences, negative regulators,or tissue-specific silencers may be inserted to reduce non-specificexpression of binding domain-immunoglobulin fusion protein encodinggenes in certain situations, such as, for example, a host that istransiently immunocompromised as part of a therapeutic strategy.Multiple repressor elements may be inserted in the promoter region.Repression of transcription is independent on the orientation ofrepressor elements or distance from the promoter. One type of repressorsequence is an insulator sequence. Such sequences inhibit transcription(Dunaway et al., Mol Cell Biol 17: 182-9, 1997; Gdula et al., Proc NatlAcad Sci USA 93:9378-83, 1996, Chan et al., J Virol 70: 5312-28, 1996;Scott and Geyer, EMBO J 14:6258-67, 1995; Kalos and Fournier, Mol CellBiol 15:198-207, 1995; Chung et al., Cell 74: 505-14, 1993) and willsilence background transcription.

Repressor elements have also been identified in the promoter regions ofthe genes for type II (cartilage) collagen, choline acetyltransferase,albumin (Hu et al., J. Cell Growth Differ. 3(9):577-588, 1992),phosphoglycerate kinase (PGK-2) (Misuno et al., Gene 119(2):293-297,1992), and in the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatasegene. (Lemaigre et al., Mol. Cell Biol. 11(2):1099-1106.) Furthermore,the negative regulatory element Tse-1 has been identified in a number ofliver specific genes, and has been shown to block cAMP response element-(CRE) mediated induction of gene activation in hepatocytes. (Boshart etal., Cell 61(5):905-916, 1990).

In preferred embodiments, elements that increase the expression of thedesired product are incorporated into the construct. Such elementsinclude internal ribosome binding sites (IRES; Wang and Siddiqui, Curr.Top. Microbiol. Immunol 203:99, 1995; Ehrenfeld and Semler, Curr. Top.Microbiol. Immunol. 203:65, 1995; Rees et al., Biotechniques 20:102,1996; Sugimoto et al., Biotechnology 12:694, 1994). IRES increasetranslation efficiency. As well, other sequences may enhance expression.For some genes, sequences especially at the 5′ end inhibit transcriptionand/or translation. These sequences are usually palindromes that canform hairpin structures. Any such sequences in the nucleic acid to bedelivered are generally deleted. Expression levels of the transcript ortranslated product are assayed to confirm or ascertain which sequencesaffect expression. Transcript levels may be assayed by any known method,including Northern blot hybridization, RNase probe protection and thelike. Protein levels may be assayed by any known method, includingELISA, western blot, immunocytochemistry or other well known techniques.

Other elements may be incorporated into the bindingdomain-immunoglobulin fusion protein encoding constructs of the presentinvention. In preferred embodiments, the construct includes atranscription terminator sequence, including a polyadenylation sequence,splice donor and acceptor sites, and an enhancer. Other elements usefulfor expression and maintenance of the construct in mammalian cells orother eukaryotic cells may also be incorporated (e.g., origin ofreplication). Because the constructs are conveniently produced inbacterial cells, elements that are necessary for, or that enhance,propagation in bacteria are incorporated. Such elements include anorigin of replication, a selectable marker and the like.

As provided herein, an additional level of controlling the expression ofnucleic acids encoding binding domain-immunoglobulin fusion proteinsdelivered to cells using the constructs of the invention may be providedby simultaneously delivering two or more differentially regulatednucleic acid constructs. The use of such a multiple nucleic acidconstruct approach may permit coordinated regulation of an immuneresponse such as, for example, spatiotemporal coordination that dependson the cell type and/or presence of another expressed encoded component.Those familiar with the art will appreciate that multiple levels ofregulated gene expression may be achieved in a similar manner byselection of suitable regulatory sequences, including but not limited topromoters, enhancers and other well known gene regulatory elements.

The present invention also relates to vectors, and to constructsprepared from known vectors that include nucleic acids of the presentinvention, and in particular to “recombinant expression constructs” thatinclude any nucleic acids encoding binding domain-immunoglobulin fusionproteins and polypeptides according to the invention as provided above;to host cells which are genetically engineered with vectors and/orconstructs of the invention and to methods of administering expressionconstructs comprising nucleic acid sequences encoding such bindingdomain-immunoglobulin fusion polypeptides and fusion proteins of theinvention, or fragments or variants thereof, by recombinant techniques.Binding domain-immunoglobulin fusion proteins can be expressed invirtually any host cell under the control of appropriate promoters,depending on the nature of the construct (e.g., type of promoter, asdescribed above), and on the nature of the desired host cell (e.g.,whether postmitotic terminally differentiated or actively dividing;e.g., whether the expression construct occurs in host cell as an episomeor is integrated into host cell genome). Appropriate cloning andexpression vectors for use with prokaryotic and eukaryotic hosts aredescribed by Sambrook, et al., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor, N.Y., (1989); as noted above, inparticularly preferred embodiments of the invention, recombinantexpression is conducted in mammalian cells that have been transfected ortransformed with the subject invention recombinant expression construct.

Typically, the constructs are derived from plasmid vectors. A preferredconstruct is a modified pNASS vector (Clontech, Palo Alto, Calif.),which has nucleic acid sequences encoding an ampicillin resistance gene,a polyadenylation signal and a T7 promoter site. Other suitablemammalian expression vectors are well known (see, e.g., Ausubel et al.,1995; Sambrook et al., supra; see also, e.g., catalogues fromInvitrogen, San Diego, Calif.; Novagen, Madison, Wis.; Pharmacia,Piscataway, N.J.; and others). Presently preferred constructs may beprepared that include a dihydrofolate reductase (DHFR) encoding sequenceunder suitable regulatory control, for promoting enhanced productionlevels of the binding domain-immunoglobulin fusion protei, which levelsresult from gene amplification following application of an appropriateselection agent (e.g., methetrexate).

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence, as described above.The heterologous structural sequence is assembled in appropriate phasewith translation initiation and termination sequences. Thus, forexample, the binding domain-immunoglobulin fusion protein encodingnucleic acids as provided herein may be included in any one of a varietyof expression vector constructs as a recombinant expression constructfor expressing a binding domain-immunoglobulin fusion polypeptide in ahost cell. In certain preferred embodiments the constructs are includedin formulations that are administered in vivo. Such vectors andconstructs include chromosomal, nonchromosomal and synthetic DNAsequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA;yeast plasmids; vectors derived from combinations of plasmids and phageDNA, viral DNA, such as vaccinia, adenovirus, fowl pox virus, andpseudorabies, or replication deficient retroviruses as described below.However, any other vector may be used for preparation of a recombinantexpression construct, and in preferred embodiments such a vector will bereplicable and viable in the host.

The appropriate DNA sequence(s) may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Standard techniques for cloning, DNA isolation, amplification andpurification, for enzymatic reactions involving DNA ligase, DNApolymerase, restriction endonucleases and the like, and variousseparation techniques are those known and commonly employed by thoseskilled in the art. A number of standard techniques are described, forexample, in Ausubel et al. (1993 Current Protocols in Molecular Biology,Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc., Boston, Mass.);Sambrook et al. (1989 Molecular Cloning, Second Ed., Cold Spring HarborLaboratory, Plainview, N.Y.); Maniatis et al. (1982 Molecular Cloning,Cold Spring Harbor Laboratory, Plainview, N.Y.); Glover (Ed.) (1985 DNACloning Vol. I and II, IRL Press, Oxford, UK); Hames and Higgins (Eds.),(1985 Nucleic Acid Hybridization, IRL Press, Oxford, UK); and elsewhere.

The DNA sequence in the expression vector is operatively linked to atleast one appropriate expression control sequences (e.g., a constitutivepromoter or a regulated promoter) to direct mRNA synthesis.Representative examples of such expression control sequences includepromoters of eukaryotic cells or their viruses, as described above.Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art, and preparation ofcertain particularly preferred recombinant expression constructscomprising at least one promoter or regulated promoter operably linkedto a nucleic acid encoding an binding domain-immunoglobulin fusionpolypeptide is described herein.

Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes may be increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act on a promoter to increase itstranscription. Examples including the SV40 enhancer on the late side ofthe replication origin bp 100 to 270, a cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

As provided herein, in certain embodiments the vector may be a viralvector such as a retroviral vector. (Miller et al., 1989 BioTechniques7:980; Coffin and Varmus, 1996 Retroviruses, Cold Spring HarborLaboratory Press, NY.) For example, retroviruses from which theretroviral plasmid vectors may be derived include, but are not limitedto, Moloney Murine Leukemia Virus, spleen necrosis virus, retrovirusessuch as Rous Sarcoma Virus, Harvey Sarcoma virus, avian leukosis virus,gibbon ape leukemia virus, human immunodeficiency virus, adenovirus,Myeloproliferative Sarcoma Virus, and mammary tumor virus.

Retroviruses are RNA viruses which can replicate and integrate into thegenome of a host cell via a DNA intermediate. This DNA intermediate, orprovirus, may be stably integrated into the host cell DNA. According tocertain embodiments of the present invention, an expression constructmay comprise a retrovirus into which a foreign gene that encodes aforeign protein is incorporated in place of normal retroviral RNA. Whenretroviral RNA enters a host cell coincident with infection, the foreigngene is also introduced into the cell, and may then be integrated intohost cell DNA as if it were part of the retroviral genome. Expression ofthis foreign gene within the host results in expression of the foreignprotein.

Most retroviral vector systems which have been developed for genetherapy are based on murine retroviruses. Such retroviruses exist in twoforms, as free viral particles referred to as virions, or as provirusesintegrated into host cell DNA. The virion form of the virus contains thestructural and enzymatic proteins of the retrovirus (including theenzyme reverse transcriptase), two RNA copies of the viral genome, andportions of the source cell plasma membrane containing viral envelopeglycoprotein. The retroviral genome is organized into four main regions:the Long Terminal Repeat (LTR), which contains cis-acting elementsnecessary for the initiation and termination of transcription and issituated both 5′ and 3′ of the coding genes, and the three coding genesgag, pol, and env. These three genes gag, pol, and env encode,respectively, internal viral structures, enzymatic proteins (such asintegrase), and the envelope glycoprotein (designated gp70 and p15e)which confers infectivity and host range specificity of the virus, aswell as the “R” peptide of undetermined function.

Separate packaging cell lines and vector producing cell lines have beendeveloped because of safety concerns regarding the uses of retroviruses,including their use in expression constructs as provided by the presentinvention. Briefly, this methodology employs the use of two components,a retroviral vector and a packaging cell line (PCL). The retroviralvector contains long terminal repeats (LTRs), the foreign DNA to betransferred and a packaging sequence (y). This retroviral vector willnot reproduce by itself because the genes which encode structural andenvelope proteins are not included within the vector genome. The PCLcontains genes encoding the gag, pol, and env proteins, but does notcontain the packaging signal “y”. Thus, a PCL can only form empty virionparticles by itself. Within this general method, the retroviral vectoris introduced into the PCL, thereby creating a vector-producing cellline (VCL). This VCL manufactures virion particles containing only theretroviral vector's (foreign) genome, and therefore has previously beenconsidered to be a safe retrovirus vector for therapeutic use.

“Retroviral vector construct” refers to an assembly which is, withinpreferred embodiments of the invention, capable of directing theexpression of a sequence(s) or gene(s) of interest, such as bindingdomain-immunoglobulin fusion encoding nucleic acid sequences. Briefly,the retroviral vector construct must include a 5′ LTR, a tRNA bindingsite, a packaging signal, an origin of second strand DNA synthesis and a3′ LTR. A wide variety of heterologous sequences may be included withinthe vector construct, including for example, sequences which encode aprotein (e.g., cytotoxic protein, disease-associated antigen, immuneaccessory molecule, or replacement gene), or which are useful as amolecule itself (e.g., as a ribozyme or antisense sequence).

Retroviral vector constructs of the present invention may be readilyconstructed from a wide variety of retroviruses, including for example,B, C, and D type retroviruses as well as spumaviruses and lentiviruses(see, e.g., RNA Tumor Viruses, Second Edition, Cold Spring HarborLaboratory, 1985). Such retroviruses may be readily obtained fromdepositories or collections such as the American Type Culture Collection(“ATCC”; Rockville, Md.), or isolated from known sources using commonlyavailable techniques. Any of the above retroviruses may be readilyutilized in order to assemble or construct retroviral vector constructs,packaging cells, or producer cells of the present invention given thedisclosure provided herein, and standard recombinant techniques (e.g.,Sambrook et al, Molecular Cloning: A Laboratory Manual, 2d ed., ColdSpring Harbor Laboratory Press, 1989; Kunkle, PNAS 82:488, 1985).

Suitable promoters for use in viral vectors generally may include, butare not limited to, the retroviral LTR; the SV40 promoter; and the humancytomegalovirus (CMV) promoter described in Miller, et al.,Biotechniques 7:980-990 (1989), or any other promoter (e.g., cellularpromoters such as eukaryotic cellular promoters including, but notlimited to, the histone, pol III, and β-actin promoters). Other viralpromoters which may be employed include, but are not limited to,adenovirus promoters, thymidine kinase (TK) promoters, and B19parvovirus promoters. The selection of a suitable promoter will beapparent to those skilled in the art from the teachings containedherein, and may be from among either regulated promoters or promoters asdescribed above.

As described above, the retroviral plasmid vector is employed totransduce packaging cell lines to form producer cell lines. Examples ofpackaging cells which may be transfected include, but are not limitedto, the PE501, PA317, ψ-2, ψ-AM, PA12, T19-14X, VT-19-17-H2, ψCRE,ψCRIP, GP+E-86, GP+envAm12, and DAN cell lines as described in Miller,Human Gene Therapy, 1:5-14 (1990). The vector may transduce thepackaging cells through any means known in the art. Such means include,but are not limited to, electroporation, the use of liposomes, and CaPO₄precipitation. In one alternative, the retroviral plasmid vector may beencapsulated into a liposome, or coupled to a lipid, and thenadministered to a host.

The producer cell line generates infectious retroviral vector particleswhich include the nucleic acid sequence(s) encoding the bindingdomain-immunoglobulin fusion polypeptides or fusion proteins. Suchretroviral vector particles then may be employed, to transduceeukaryotic cells, either in vitro or in vivo. The transduced eukaryoticcells will express the nucleic acid sequence(s) encoding the bindingdomain-immunoglobulin fusion polypeptide or fusion protein. Eukaryoticcells which may be transduced include, but are not limited to, embryonicstem cells, as well as hematopoietic stem cells, hepatocytes,fibroblasts, circulating peripheral blood mononuclear andpolymorphonuclear cells including myelomonocytic cells, lymphocytes,myoblasts, tissue macrophages, dendritic cells, Kupffer cells, lymphoidand reticuloendothelia cells of the lymph nodes and spleen,keratinocytes, endothelial cells, and bronchial epithelial cells.

As another example of an embodiment of the invention in which a viralvector is used to prepare the recombinant binding domain-immunoglobulinfusion expression construct, in one preferred embodiment, host cellstransduced by a recombinant viral construct directing the expression ofbinding domain-immunoglobulin fusion polypeptides or fusion proteins mayproduce viral particles containing expressed bindingdomain-immunoglobulin fusion polypeptides or fusion proteins that arederived from portions of a host cell membrane incorporated by the viralparticles during viral budding.

In another aspect, the present invention relates to host cellscontaining the above described recombinant binding domain-immunoglobulinfusion expression constructs. Host cells are genetically engineered(transduced, transformed or transfected) with the vectors and/orexpression constructs of this invention which may be, for example, acloning vector, a shuttle vector or an expression construct. The vectoror construct may be, for example, in the form of a plasmid, a viralparticle, a phage, etc. The engineered host cells can be cultured inconventional nutrient media modified as appropriate for activatingpromoters, selecting transformants or amplifying particular genes suchas genes encoding binding domain-immunoglobulin fusion polypeptides orbinding domain-immunoglobulin fusion proteins. The culture conditionsfor particular host cells selected for expression, such as temperature,pH and the like, will be readily apparent to the ordinarily skilledartisan.

The host cell can be a higher eukaryotic cell, such as a mammalian cell,or a lower eukaryotic cell, such as a yeast cell, or the host cell canbe a prokaryotic cell, such as a bacterial cell. Representative examplesof appropriate host cells according to the present invention include,but need not be limited to, bacterial cells, such as E. coli,Streptomyces, Salmonella typhimurium; fungal cells, such as yeast;insect cells, such as Drosophila S2 and Spodoptera Sf9; animal cells,such as CHO, COS or 293 cells; adenoviruses; plant cells, or anysuitable cell already adapted to in vitro propagation or so establishedde novo. The selection of an appropriate host is deemed to be within thescope of those skilled in the art from the teachings herein.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell23:175 (1981), and other cell lines capable of expressing a compatiblevector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines.Mammalian expression vectors will comprise an origin of replication, asuitable promoter and enhancer, and also any necessary ribosome bindingsites, polyadenylation site, splice donor and acceptor sites,transcriptional termination sequences, and 5′ flanking nontranscribedsequences, for example as described herein regarding the preparation ofbinding domain-immunoglobulin fusion expression constructs. DNAsequences derived from the SV40 splice, and polyadenylation sites may beused to provide the required nontranscribed genetic elements.Introduction of the construct into the host cell can be effected by avariety of methods with which those skilled in the art will be familiar,including but not limited to, for example, calcium phosphatetransfection, DEAE-Dextran mediated transfection, or electroporation(Davis et al., 1986 Basic Methods in Molecular Biology).

The present invention binding domain-immunoglobulin fusion proteins maybe formulated into pharmaceutical compositions for administrationaccording to well known methodologies. Pharmaceutical compositionsgenerally comprise one or more recombinant expression constructs, and/orexpression products of such constructs, in combination with apharmaceutically acceptable carrier, excipient or diluent. Such carrierswill be nontoxic to recipients at the dosages and concentrationsemployed. For nucleic acid-based formulations, or for formulationscomprising expression products of the subject invention recombinantconstructs, about 0.01 μg/kg to about 100 mg/kg body weight will beadministered, typically by the intradermal, subcutaneous, intramuscularor intravenous route, or by other routes. A preferred dosage is about 1μg/kg to about 1 mg/kg, with about 5 μg/kg to about 200 μg/kgparticularly preferred. It will be evident to those skilled in the artthat the number and frequency of administration will be dependent uponthe response of the host. “Pharmaceutically acceptable carriers” fortherapeutic use are well known in the pharmaceutical art, and aredescribed, for example, in Remingtons Pharmaceutical Sciences, MackPublishing Co. (A. R. Gennaro edit. 1985). For example, sterile salineand phosphate-buffered saline at physiological pH may be used.Preservatives, stabilizers, dyes and even flavoring agents may beprovided in the pharmaceutical composition. For example, sodiumbenzoate, sorbic acid and esters of p-hydroxybenzoic acid may be addedas preservatives. Id. at 1449. In addition, antioxidants and suspendingagents may be used. Id.

“Pharmaceutically acceptable salt” refers to salts of the compounds ofthe present invention derived from the combination of such compounds andan organic or inorganic acid (acid addition salts) or an organic orinorganic base (base addition salts). The compounds of the presentinvention may be used in either the free base or salt forms, with bothforms being considered as being within the scope of the presentinvention.

The pharmaceutical compositions that contain one or more bindingdomain-immunoglobulin fusion protein encoding constructs (or theirexpressed products) may be in any form which allows for the compositionto be administered to a patient. For example, the composition may be inthe form of a solid, liquid or gas (aerosol). Typical routes ofadministration include, without limitation, oral, topical, parenteral(e.g., sublingually or buccally), sublingual, rectal, vaginal, andintranasal. The term parenteral as used herein includes subcutaneousinjections, intravenous, intramuscular, intrasternal, intracavernous,intrathecal, intrameatal, intraurethral injection or infusiontechniques. The pharmaceutical composition is formulated so as to allowthe active ingredients contained therein to be bioavailable uponadministration of the composition to a patient. Compositions that willbe administered to a patient take the form of one or more dosage units,where for example, a tablet may be a single dosage unit, and a containerof one or more compounds of the invention in aerosol form may hold aplurality of dosage units.

For oral administration, an excipient and/or binder may be present.Examples are sucrose, kaolin, glycerin, starch dextrins, sodiumalginate, carboxymethylcellulose and ethyl cellulose. Coloring and/orflavoring agents may be present. A coating shell may be employed.

The composition may be in the form of a liquid, e.g., an elixir, syrup,solution, emulsion or suspension. The liquid may be for oraladministration or for delivery by injection, as two examples. Whenintended for oral administration, preferred compositions contain, inaddition to one or more binding domain-immunoglobulin fusion constructor expressed product, one or more of a sweetening agent, preservatives,dye/colorant and flavor enhancer. In a composition intended to beadministered by injection, one or more of a surfactant, preservative,wetting agent, dispersing agent, suspending agent, buffer, stabilizerand isotonic agent may be included.

A liquid pharmaceutical composition as used herein, whether in the formof a solution, suspension or other like form, may include one or more ofthe following adjuvants: sterile diluents such as water for injection,saline solution, preferably physiological saline, Ringer's solution,isotonic sodium chloride, fixed oils such as synthetic mono ordigylcerides which may serve as the solvent or suspending medium,polyethylene glycols, glycerin, propylene glycol or other solvents;antibacterial agents such as benzyl alcohol or methyl paraben;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. The parenteral preparation can be enclosedin ampoules, disposable syringes or multiple dose vials made of glass orplastic. Physiological saline is a preferred adjuvant. An injectablepharmaceutical composition is preferably sterile.

It may also be desirable to include other components in the preparation,such as delivery vehicles including but not limited to aluminum salts,water-in-oil emulsions, biodegradable oil vehicles, oil-in-wateremulsions, biodegradable microcapsules, and liposomes. Examples ofimmunostimulatory substances (adjuvants) for use in such vehiclesinclude N-acetylmuramyl-L-alanine-D-isoglutamine (MDP),lipopoly-saccharides (LPS), glucan, IL-12, GM-CSF, gamma interferon andIL-15.

While any suitable carrier known to those of ordinary skill in the artmay be employed in the pharmaceutical compositions of this invention,the type of carrier will vary depending on the mode of administrationand whether a sustained release is desired. For parenteraladministration, such as subcutaneous injection, the carrier preferablycomprises water, saline, alcohol, a fat, a wax or a buffer. For oraladministration, any of the above carriers or a solid carrier, such asmannitol, lactose, starch, magnesium stearate, sodium saccharine,talcum, cellulose, glucose, sucrose, and magnesium carbonate, may beemployed. Biodegradable microspheres (e.g., polylactic galactide) mayalso be employed as carriers for the pharmaceutical compositions of thisinvention. Suitable biodegradable microspheres are disclosed, forexample, in U.S. Pat. Nos. 4,897,268 and 5,075,109. In this regard, itis preferable that the microsphere be larger than approximately 25microns.

Pharmaceutical compositions may also contain diluents such as buffers,antioxidants such as ascorbic acid, low molecular weight (less thanabout 10 residues) polypeptides, proteins, amino acids, carbohydratesincluding glucose, sucrose or dextrins, chelating agents such as EDTA,glutathione and other stabilizers and excipients. Neutral bufferedsaline or saline mixed with nonspecific serum albumin are exemplaryappropriate diluents. Preferably, product is formulated as alyophilizate using appropriate excipient solutions (e.g., sucrose) asdiluents.

As described above, the subject invention includes compositions capableof delivering nucleic acid molecules encoding bindingdomain-immunoglobulin fusion proteins. Such compositions includerecombinant viral vectors (e.g., retroviruses (see WO 90/07936, WO91/02805, WO 93/25234, WO 93/25698, and WO 94/03622), adenovirus (seeBerkner, Biotechniques 6:616-627, 1988; Li et al., Hum. Gene Ther.4:403-409, 1993; Vincent et al., Nat. Genet. 5:130-134, 1993; and Kollset al., Proc. Natl. Acad. Sci. USA 91:215-219, 1994), pox virus (seeU.S. Pat. No. 4,769,330; U.S. Pat. No. 5,017,487; and WO 89/01973)),recombinant expression construct nucleic acid molecules complexed to apolycationic molecule (see WO 93/03709), and nucleic acids associatedwith liposomes (see Wang et al., Proc. Natl. Acad. Sci. USA 84:7851,1987). In certain embodiments, the DNA may be linked to killed orinactivated adenovirus (see Curiel et al., Hum. Gene Ther. 3:147-154,1992; Cotton et al., Proc. Natl. Acad. Sci. USA 89:6094, 1992). Othersuitable compositions include DNA-ligand (see Wu et al., J. Biol. Chem.264:16985-16987, 1989) and lipid-DNA combinations (see Felgner et al.,Proc. Natl. Acad. Sci. USA 84:7413-7417, 1989).

In addition to direct in vivo procedures, ex vivo procedures may be usedin which cells are removed from a host, modified, and placed into thesame or another host animal. It will be evident that one can utilize anyof the compositions noted above for introduction of bindingdomain-immunoglobulin fusion proteins or of bindingdomain-immunoglobulin fusion protein encoding nucleic acid moleculesinto tissue cells in an ex vivo context. Protocols for viral, physicaland chemical methods of uptake are well known in the art.

Accordingly, the present invention is useful for treating a patienthaving a B-cell disorder or a malignant condition, or for treating acell culture derived from such a patient. As used herein, the term“patient” refers to any warm-blooded animal, preferably a human. Apatient may be afflicted with cancer, such as B-cell lymphoma, or may benormal (i.e., free of detectable disease and infection). A “cellculture” is any preparation amenable to ex vivo treatment, for example apreparation containing immunocompetent cells or isolated cells of theimmune system (including, but not limited to, T cells, macrophages,monocytes, B cells and dendritic cells). Such cells may be isolated byany of a variety of techniques well known to those of ordinary skill inthe art (e.g., Ficoll-hypaque density centrifugation). The cells may(but need not) have been isolated from a patient afflicted with a B-celldisorder or a malignancy, and may be reintroduced into a patient aftertreatment.

A liquid composition intended for either parenteral or oraladministration should contain an amount of binding domain-immunoglobulinfusion protein encoding construct or expressed product such that asuitable dosage will be obtained. Typically, this amount is at least0.01 wt % of a binding domain-immunoglobulin fusion construct orexpressed product in the composition. When intended for oraladministration, this amount may be varied to be between 0.1 and about70% of the weight of the composition. Preferred oral compositionscontain between about 4% and about 50% of binding domain-immunoglobulinfusion construct or expressed product(s). Preferred compositions andpreparations are prepared so that a parenteral dosage unit containsbetween 0.01 to 1% by weight of active compound.

The pharmaceutical composition may be intended for topicaladministration, in which case the carrier may suitably comprise asolution, emulsion, ointment or gel base. The base, for example, maycomprise one or more of the following: petrolatum, lanolin, polyethyleneglycols, beeswax, mineral oil, diluents such as water and alcohol, andemulsifiers and stabilizers. Thickening agents may be present in apharmaceutical composition for topical administration. If intended fortransdermal administration, the composition may include a transdermalpatch or iontophoresis device. Topical formulations may contain aconcentration of the binding domain-immunoglobulin fusion construct orexpressed product of from about 0.1 to about 10% w/v (weight per unitvolume).

The composition may be intended for rectal administration, in the form,e.g., of a suppository which will melt in the rectum and release thedrug. The composition for rectal administration may contain anoleaginous base as a suitable nonirritating excipient. Such basesinclude, without limitation, lanolin, cocoa butter and polyethyleneglycol.

In the methods of the invention, the binding domain-immunoglobulinfusion encoding constructs or expressed product(s) may be administeredthrough use of insert(s), bead(s), timed-release formulation(s),patch(es) or fast-release formulation(s).

The following Examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1 Cloning of the 2H7 Variable Regions and Constructionand Sequencing of 2H7scFv-Ig

This Example illustrates the cloning of cDNA molecules that encode theheavy chain and light chain variable regions of the monoclonal antibody2H7. This Example also demonstrates the construction, sequencing, andexpression of 2H7scFv-Ig.

Hybridoma cells expressing 2H7 monoclonal antibody that specificallybound to CD20 were provided by Ed Clark at the University of Washington,Seattle, Wash. Prior to harvesting, hybridoma cells were kept in logphase growth for several days in RPMI 1640 media (Life Technologies,Gaithersburg, Md.) supplemented with glutamine, pyruvate, DMEMnon-essential amino acids, and penicillin-streptomycin. Cells werepelleted by centrifugation from the culture medium, and 2×10⁷ cells wereused to prepare RNA. RNA was isolated from the 2H7-producing hybridomacells using the Pharmingen (San Diego, Calif.) total RNA isolation kit(Catalog # 45520K) according to the manufacturer's instructionsaccompanying the kit. One microgram (1 μg) of total RNA was used astemplate to prepare cDNA by reverse transcription. The RNA and 300 ngrandom primers were combined and denatured at 72° C. for 10 minutesprior to addition of enzyme. Superscript II reverse transcriptase (LifeTechnologies) was added to the RNA plus primer mixture in a total volumeof 25 μl in the presence of 5× second strand buffer and 0.1 M DTTprovided with the enzyme. The reverse transcription reaction was allowedto proceed at 42° C. for one hour.

The 2H7 cDNA generated in the randomly primed reverse transcriptasereaction and V region specific primers were used to amplify by PCR thevariable regions for the light and heavy chain of the 2H7 antibody. TheV region specific primers were designed using the published sequence(Genbank accession numbers M17954 for V_(L) and M17953 for V_(H)) as aguide. The two variable chains were designed with compatible endsequences so that an scFv could be assembled by ligation of the two Vregions after amplification and restriction enzyme digestion.

A (gly₄ser)₃ peptide linker to be inserted between the two V regions wasincorporated by adding the extra nucleotides to the antisense primer forthe V_(L) of 2H7. A Sac I restriction site was also introduced at thejunction between the two V regions. The sense primer used to amplify the2H7 V_(L), that included a HindIII restriction site and the light chainleader peptide was 5′-gtc aag ctt gcc gcc atg gat ttt caa gtg cag attttt cag c-3′ (SEQ ID NO:23). The antisense primer was 5′-gtc gtc gagctccca cct cct cca gat cca cca ccg ccc gag cca ccg cca cct ttc agc tccagc ttg gtc cc-3′ (SEQ ID NO:24). The reading frame of the V region isindicated as a bold, underlined codon. The Hind III and SacI sites areindicated by underlined italicized sequences.

The V_(H) domain was amplified without a leader peptide, but included a5′ SacI restriction site for fusion to the V_(L) and a BclI restrictionsite at the 3′ end for fusion to various tails, including the human IgG1Fc domain and the truncated forms of CD40 ligand, CD154. The senseprimer was 5′-gct gct gag ctc tca ggc tta tct aca gca agt ctg g-3′ (SEQID NO:25). The SacI site is indicated in italicized and underlined font,and the reading frame of the codon for the first amino acid of the V_(H)domain is indicated in bold, underlined type. The antisense primer was5′-gtt gtc tga tca gag acg gtg acc gtg gtc cc-3′ (SEQ ID NO:26). TheBclI site is indicated in italicized, underlined type, and the lastserine of the V_(H) domain sequence is indicated in bold, underlinedtype.

The scFv-Ig was assembled by inserting the 2H7 scFv HindIII-BclIfragment into pUC19 containing the human IgG1 hinge, CH2, and CH3regions, which was digested with restriction enzymes, HindIII and BclI.After ligation, the ligation products were transformed into DH5αbacteria. Positive clones were screened for the properly insertedfragments using the SacI site at the V_(L)-V_(H) junction of 2H7 as adiagnostic site. The 2H7scFv-Ig cDNA was subjected to cycle sequencingon a PE 9700 thermocycler using a 25-cycle program by denaturing at 96°C. for 10 seconds, annealing at 50° C. for 30 seconds, and extending at72° C. for 4 minutes. The sequencing primers were pUC forward andreverse primers and an internal primer that annealed to the CH2 domainhuman in the IgG constant region portion. Sequencing reactions wereperformed using the Big Dye Terminator Ready Sequencing Mix (PE-AppliedBiosystems, Foster City, Calif.) according to the manufacturer'sinstructions. Samples were subsequently purified using Centrisep columns(Catalog # CS-901, Princeton Separations, Adelphia, N.J.), the eluatesdried in a Savant vacuum dryer, denatured in Template SuppressionReagent (PE-ABI), and analyzed on an ABI 310 Genetic Analyzer(PE-Applied Biosystems). The sequence was edited, translated, andanalyzed using Vector Nti version 6.0 (Informax, North Bethesda, Md.).FIG. 1 shows the cDNA and predicted amino acid sequence of the2H7scFv-Ig construct.

Example 2 Expression of 2H7 ScFv-Ig in Stable CHO Cell Lines

This Example illustrates expression of 2H7scFv-Ig in a eukaryotic cellline and characterization of the expressed 2H7scFv-Ig by SDS-PAGE and byfunctional assays, including ADCC and complement fixation.

The 2H7scFv-Ig HindIII-XbaI (˜1.6 kb) fragment with correct sequence wasinserted into the mammalian expression vector pD18, and DNA frompositive clones was amplified using QIAGEN plasmid preparation kits(QIAGEN, Valencia, Calif.). The recombinant plasmid DNA (100 μg) wasthen linearized in a nonessential region by digestion with AscI,purified by phenol extraction, and resuspended in tissue culture media,Excell 302 (Catalog # 14312-79P, JRH Biosciences, Lenexa, Kans.). Cellsfor transfection, CHO DG44 cells, were kept in logarithmic growth, and10⁷ cells harvested for each transfection reaction. Linearized DNA wasadded to the CHO cells in a total volume of 0.8 ml for electroporation.

Stable production of the 2H7 scFv-Ig fusion protein (SEQ. ID NO:15) wasachieved by electroporation of a selectable, amplifiable plasmid, pD18,containing the 2H7 scFv-Ig cDNA under the control of the CMV promoter,into Chinese Hamster Ovary (CHO) cells (all cell lines from AmericanType Culture Collection, Manassas, Va., unless otherwise noted). The 2H7expression cassette was subcloned downstream of the CMV promoter intothe vector multiple cloning site as a ˜1.6 kb HindIII-XbaI fragment. ThepD18 vector is a modified version of pcDNA3 encoding the DHFR selectablemarker with an attenuated promoter to increase selection pressure forthe plasmid. Plasmid DNA was prepared using Qiagen maxiprep kits, andpurified plasmid was linearized at a unique AscI site prior to phenolextraction and ethanol precipitation. Salmon sperm DNA (Sigma-Aldrich,St. Louis, Mo.) was added as carrier DNA, and 100 μg each of plasmid andcarrier DNA was used to transfect 10⁷ CHO DG44 cells by electroporation.Cells were grown to logarithmic phase in Excell 302 media (JRHBiosciences) containing glutamine (4 mM), pyruvate, recombinant insulin,penicillin-streptomycin, and 2×DMEM nonessential amino acids (all fromLife Technologies, Gaithersburg, Md.), hereafter referred to as “Excell302 complete” media. Media for untransfected cells also contained HT(diluted from a 100× solution of hypoxanthine and thymidine) (LifeTechnologies). Media for transfections under selection contained varyinglevels of methotrexate (Sigma-Aldrich) as selective agent, ranging from50 nM to 5 μM. Electroporations were performed at 275 volts, 950 μF.Transfected cells were allowed to recover overnight in non-selectivemedia prior to selective plating in 96 well flat bottom plates (Costar)at varying serial dilutions ranging from 125 cells/well to 2000cells/well. Culture media for cell cloning was Excell 302 complete,containing 100 nM methotrexate. Once clonal outgrowth was sufficient,serial dilutions of culture supernatants from master wells were screenedfor binding to CD20-CHO transfected cells. The clones with the highestproduction of the fusion protein were expanded into T25 and then T75flasks to provide adequate numbers of cells for freezing and for scalingup production of the 2H7scFvIg. Production levels were further increasedin cultures from three clones by progressive amplification inmethotrexate containing culture media. At each successive passage ofcells, the Excell 302 complete media contained an increasedconcentration of methotrexate, such that only the cells that amplifiedthe DHFR plasmid could survive.

Supernatants were collected from CHO cells expressing the 2H7scFv-Ig,filtered through 0.2 μm PES express filters (Nalgene, Rochester, N.Y.)and were passed over a Protein A-agarose (IPA 300 crosslinked agarose)column (Repligen, Needham, Mass.). The column was washed with PBS, andthen bound protein was eluted using 0.1 M citrate buffer, pH 3.0.Fractions were collected and eluted protein was neutralized using 1MTris, pH 8.0, prior to dialysis overnight in PBS. Concentration of thepurified 2H7scFv-Ig (SEQ ID NO:15) was determined by absorption at 280nm. An extinction coefficient of 1.77 was determined using the proteinanalysis tools in the Vector Nti Version 6.0 Software package (Informax,North Bethesda, Md.). This program uses the amino acid composition datato calculate extinction coefficients.

Production levels of 2H7scFv-Ig by transfected, stable CHO cells wereanalyzed by flow cytometry. Purified 2H7scFv-Ig to CHO cells was allowedto bind to CHO cells that expressed CD20 (CD20 CHO) and analyzed by flowcytometry using a fluorescein-conjugated anti-human IgG second stepreagent (Catalog Numbers H10101 and H10501, CalTag, Burlingame, Calif.).FIG. 2 (top) shows a standard curve generated by titration of 2H7scFv-Igbinding to CD20 CHO. At each concentration of 2H7scFv-Ig, the meanbrightness of the fluorescein signal in linear units is shown.Supernatants collected from T flasks containing stable CHO cell clonesexpressing 2H7scFv-Ig were then allowed to bind to CD20 CHO and thebinding was analyzed by flow cytometry. The fluorescein signal generatedby 2H7scFv-Ig contained in the supernatants was measured and the2H7scFv-Ig concentration in the supernatants was calculated from thestandard curve (FIG. 2, bottom).

Purified 2H7scFv-Ig (SEQ ID NO:15) was analyzed by electrophoresis onSDS-Polyacrylamide gels. Samples of 2H7scFv-Ig, purified by independentProtein A Agarose column runs, were boiled in SDS sample buffer withoutreduction of disulfide bonds and applied to SDS 10% Tris-BIS gels(Catalog # NP0301, Novex, Carlsbad, Calif.). Twenty micrograms of eachpurified batch was loaded on the gels. The proteins were visualizedafter electrophoresis by Coomassie Blue staining (Pierce Gel Code BlueStain Reagent, Catalog #24590, Pierce, Rockford, Ill.), and destainingin distilled water. Molecular weight markers were included on the samegel (Kaleidoscope Prestained Standards, Catalog # 161-0324, Bio-Rad,Hercules, Calif.). The results are presented in FIG. 3. The numbersabove the lanes designate independent purification batches. Themolecular weights in kilodaltons of the size markers are indicated onthe left side of the figure. Further experiments with alternative samplepreparation conditions indicated that reduction of disulfide bonds byboiling the protein in SDS sample buffer containing DTT or2-mercaptoethanol caused the 2H7scFv-Ig to aggregate.

Any number of other immunological parameters may be monitored usingroutine assays that are well known in the art. These may include, forexample, antibody dependent cell-mediated cytotoxicity (ADCC) assays,secondary in vitro antibody responses, flow immunocytofluorimetricanalysis of various peripheral blood or lymphoid mononuclear cellsubpopulations using well established marker antigen systems,immunohistochemistry or other relevant assays. These and other assaysmay be found, for example, in Rose et al. (Eds.), Manual of ClinicalLaboratory Immunology, 5^(th) Ed., 1997 American Society ofMicrobiology, Washington, D.C.

The ability of 2H7scFv-Ig to kill CD20 positive cells in the presence ofcomplement was tested using B cell lines Ramos and Bjab. Rabbitcomplement (Pel-Freez, Rogers, Ak.) was used in the assay at a finaldilution of 1/10. Purified 2H7scFv-Ig was incubated with B cells andcomplement for 45 minutes at 37° C., followed by counting of live anddead cells by trypan blue exclusion. The results in FIG. 4A show that inthe presence of rabbit complement, 2H7scFv-Ig lysed B cells expressingCD20.

The ability of 2H7scFv-Ig to kill CD20 positive cells in the presence ofperipheral blood mononuclear cells (PBMC) was tested by measuring therelease of ⁵¹Cr from labeled Bjab cells in a 4-hour assay using a 100:1ratio of PBMC to Bjab cells. The results shown in FIG. 4B indicated that2H7scFv-Ig can mediate antibody dependent cellular cytotoxicity (ADCC)because the release of ⁵¹Cr was higher in the presence of both PBMC and2H7scFv-Ig than in the presence of either PBMC or 2H7scFv-Ig alone.

Example 3 Effect of Simultaneous Ligation of CD20 and CD40 on Growth ofNormal B Cells, and on CD95 Expression, and Induction of Apoptosis

This example illustrates the effect of cross-linking of CD20 and CD40expressed on the cell surface on cell proliferation.

Dense resting B cells were isolated from human tonsil by a Percoll stepgradient and T cells were removed by E-rosetting. Proliferation ofresting, dense tonsillar B cells was measured by uptake of³[H]-thymidine during the last 12 hours of a 4-day experiment.Proliferation was measured in quadruplicate cultures with means andstandard deviations as shown. Murine anti-human CD20 mAb 1F5 (anti-CD20)was used alone or was cross-linked with anti-murine K mAb 187.1(anti-CD20XL). CD40 activation was accomplished using soluble humanCD154 fused with murine CD8 (CD154) (Hollenbaugh et al., EMBO J. 11:4212-21 (1992)), and CD40 cross-linking was accomplished usinganti-murine CD8 mAb 53-6 (CD154XL). This procedure allowed simultaneouscross-linking of CD20 and CD40 on the cell surface. The results arepresented in FIG. 5.

The effect of CD20 and CD40 cross-linking on Ramos cells, a B lymphomacell line, was examined. Ramos cells were analyzed for CD95 (Fas)expression and percent apoptosis eighteen hours after treatment (no goatanti-mouse IgG (GAM)) and/or cross-linking (+GAM) using murine mAbs thatspecifically bind CD20 (1F5) and CD40 (G28-5). Control cells weretreated with a non-binding isotype control (64.1) specific for CD3.

Treated Ramos cells were harvested, incubated with FITC-anti-CD95, andanalyzed by flow cytometry to determine the relative expression level ofFas on the cell surface after CD20 or CD40 cross-linking. Data isplotted as mean fluorescence of cells after treatment with the stimuliindicated (FIG. 6A).

Treated Ramos cells from the same experiment were harvested and bindingof annexin V was measured to indicate the percentage apoptosis in thetreated cultures. Apoptosis was measured by binding of Annexin V 18hours after cross-linking of CD20 and CD40 using 1F5 and G28-5 followedby cross-linking with GAM. Binding of Annexin V was measured using aFITC-Annexin V kit (Catalog # PN-IM2376, Immunotech, Marseille, France).Annexin V binding is known to be an early event in progression of cellsinto apoptosis. Apoptosis, or programmed cell death, is a processcharacterized by a cascade of catabolic reactions leading to cell deathby suicide. In the early phase of apoptosis, before cells changemorphology and hydrolyze DNA, the integrity of the cell membrane ismaintained but cells lose the asymmetry of their membrane phospholipids,exposing negatively charged phospholipids, such as phosphatidylserine,at the cell surface. Annexin V, a calcium and phopholipid bindingprotein, binds preferentially and with high affinity tophosphatidylserine. Results demonstrating the effect of cross-linkingboth CD20 and CD40 on expression of the FAS receptor (CD95) arepresented in FIG. 6B. The effect of cross-linking of both CD20 and CD40on Annexin V binding to cells is shown in FIG. 6B.

Example 4 Construction and Characterization of 2H7 ScFv-CD154 FusionProteins

To construct a molecule capable of binding to both CD20 and CD40, cDNAencoding the 2H7 scFv was fused with cDNA encoding CD154, the CD40ligand. The 2H7 scFv cDNA encoded on the HindIII-BclI fragment wasremoved from the 2H7 scFvIg construct, and inserted into a pD18 vectoralong with a BamHI-XbaI cDNA fragment encoding the extracellular domainof human CD154. The extracellular domain is encoded at the carboxyterminus of CD154, similar to other type II membrane proteins.

The extracellular domain of human CD154 was PCR amplified using cDNAgenerated with random primers and RNA from human T lymphocytes activatedwith PHA (phytohemagglutinin). The primer sets included two different 5′or sense primers that created fusion junctions at two differentpositions within the extracellular domain of CD154. Two different fusionjunctions were designed that resulted in a short or truncated form (formS4) including amino acids 108 (Glu)-261 (Leu)+(Glu), and a long orcomplete form (form L2) including amino acids 48 (Arg)-261 (Leu)+(Glu),of the extracellular domain of CD154, both constructed as BamHI-XbaIfragments. The sense primer which fuses the two different truncatedextracellular domains to the 2H7scFv includes a BamHI site for cloning.The sense primer for the S4 form of the CD154 cDNA is designatedSEQUENCE ID NO:27 or CD154BAM108 and encodes a 34 mer with the followingsequence: 5′-gtt gtc gga tcc aga aaa cag ctt tga aat gca a-3′, while theantisense primer is designated SEQUENCE ID NO:28 or CD154XBA and encodesa 44 mer with the following sequence: 5′-gtt gtt tct aga tta tca ctc gagttt gag taa gcc aaa gga cg-3′.

The oligonucleotide primers used in amplifying the long form (L2) of theCD154 extracellular domain encoding amino acids 48 (Arg)-261(Leu)+(Glu), were as follows: The sense primer designated CD154 BAM48(SEQUENCE ID NO:29) encoded a 35-mer with the following sequence: 5′-gttgtc gga tcc aag aag gtt gga caa gat aga ag-3′. The antisense primerdesignated CD154XBA (SEQUENCE ID NO:28) encoded the 44-mer: 5′-gtt gtttct aga tta tca ctc gag ttt gag taa gcc aaa gga cg-3′. Other PCRreaction conditions were identical to those used for amplifying the 2H7scFv (see Example 1). PCR fragments were purified by PCR quick kits(QIAGEN, San Diego, Calif.), eluted in 30 μl ddH₂O, and digested withBamHI and XbaI (Roche) restriction endonucleases in a 40 μl reactionvolume at 37° C. for 3 hours. Fragments were gel purified, purifiedusing QIAEX kits according to the manufacturer's instructions (QIAGEN),and ligated along with the 2H7 HindIII-BclI fragment into the pD18expression vector digested with HindIII+XbaI. Ligation reactions weretransformed into DH5-alpha chemically competent bacteria and plated ontoLB plates containing 100 μg/ml ampicillin. Transformants were grownovernight at 37° C., and isolated colonies used to inoculate 3 ml liquidcultures in Luria Broth containing 100 μg/ml ampicillin. Clones werescreened after mini-plasmid preparations (QIAGEN) for insertion of boththe 2H7 scFv and the CD154 extracellular domain fragments.

The 2H7scFv-CD154 construct cDNAs were subjected to cycle sequencing ona PE 9700 thermocycler using a 25-cycle program that includeddenaturating at 96° C., 10 seconds, annealing at 50° C. for 5 seconds,and extension at 60° C., for 4 minutes. The sequencing primers used werepD18 forward (SEQ ID NO:30: 5′-gtctatataagcagagctctggc-3′) and pD18reverse (SEQ ID NO:31: 5′-cgaggctgatcagcgagctctagca-3′) primers. Inaddition, an internal primer was used that had homology to the humanCD154 sequence (SEQ ID NO:32: 5′-ccgcaatttgaggattctgatcacc-3′).Sequencing reactions included primers at 3.2 pmol, approximately 200 ngDNA template, and 8 μl sequencing mix. Sequencing reactions wereperformed using the Big Dye Terminator Ready Sequencing Mix (PE-AppliedBiosystems, Foster City, Calif.) according to the manufacturer'sinstructions. Samples were subsequently purified using Centrisep columns(Princeton Separations, Adelphia, N.J.). The eluates were dried in aSavant speed-vacuum dryer, denatured in 20 μl template SuppressionReagent (ABI) at 95° C. for 2 minutes, and analyzed on an ABI 310Genetic Analyzer (PE-Applied Biosystems). The sequence was edited,translated, and analyzed using Vector Nti version 6.0 (Informax, NorthBethesda, Md.). The 2H7scFv-CD154 L2 cDNA sequence and predicted aminoacid sequence is presented in FIG. 7A, and 2H7scFv-CD154 S4 cDNAsequence and predicted amino acid sequence is presented in FIG. 7B.

The binding activity of the 2H7 scFv-CD154 fusion proteins (SEQ ID NO:33and 34) to CD20 and CD40 simultaneously was determined by flowcytometry. The assay used CHO cell targets that express CD20. After a45-minute incubation of CD20 CHO cells with supernatants from cellstransfected with the 2H7 scFv-CD154 expression plasmid, the CD20 CHOcells were washed twice and incubated with biotin-conjugated CD40-Igfusion protein in PBS/2% FBS. After 45 min, cells were washed twice andincubated with phycoerythrin (PE)-labeled strepavidin at 1:100 in PBS/2%FBS (Molecular Probes, Eugene Oreg.). After an additional 30 minincubation, cells were washed 2× and were analyzed by flow cytometry.The results show that the 2H7 scFv-CD154 molecule was able to bind toCD20 on the cell surface and to capture biotin-conjugated CD40 fromsolution (FIG. 8).

To determine the effect of the 2H7scFv-CD154 on growth and viability ofB lymphoma and lymphoblastoid cell lines, cells were incubated with2H7scFv-CD154 L2 (SEQ. ID NO:33) for 12 hours and then examined forbinding of Annexin V. Binding of Annexin V was measured using aFITC-Annexin V kit (Immunotech, Marseille, France, Catalog # PN-IM2376).B cell lines were incubated in 1 ml cultures with dilutions ofconcentrated, dialyzed supernatants from cells expressing secreted formsof the 2H7scFv-CD154 fusion proteins. The results are presented in FIG.9.

The growth rate of the Ramos B lymphoma cell line in the presence of2H7scFv-CD154 was examined by uptake of ³H-thymidine for the last 6hours of a 24-hour culture. The effect of 2H7scFv-CD154 on cellproliferation is shown in FIG. 10.

Example 5 Construction and Characterization of CytoxB AntibodyDerivatives

CytoxB antibodies were derived from the 2H7 scFv-IgG polypeptide. The2H7 scFv (see Example 1) was linked to the human IgG1 Fc domain via analtered hinge domain (see FIG. 11). Cysteine residues in the hingeregion were substituted with serine residues by site-directedmutagenesis and other methods known in the art. The mutant hinge wasfused either to a wild-type Fc domain to create one construct,designated CytoB-MHWTG1C, or was fused to a mutated Fc domain(CytoxB-MHMG1C) that had additional mutations introduced into the CH2domain. Amino acid residues in CH2 that are implicated in effectorfunction are illustrated in FIG. 11. Mutations of one or more of theseresidues may reduce FcR binding and mediation of effector functions. Inthis example, the leucine residue 234 known in the art to be importantto Fc receptor binding, was mutated in the 2H7 scFv fusion protein,CytoxB-[MG1H/MG1C]. In another construct, the human IgG1 hinge regionwas substituted with a portion of the human IgA hinge, which was fusedto wild-type human Fc domain (CytoxB-IgAHWTHG1C). (See FIG. 11). Thismutated hinge region allows expression of a mixture of monomeric anddimeric molecules that retain functional properties of the human IgG1CH2 and CH3 domains. Synthetic, recombinant cDNA expression cassettesfor these molecules were constructed and polypeptides were expressed inCHODG44 cells according to methods described in Example 2.

Purified fusion protein derivatives of CytoxB-scFvIg molecules wereanalyzed by SDS-PAGE according to the methods described in Example 2.Polyacrylamide gels were run under non-reducing and reducing conditions.Two different molecule weight marker sets, BioRad prestained markers,(BioRad, Hercules, Calif.) and Novex Multimark molecular weight markerswere loaded onto each gel. The migration patterns of the differentconstructs and of Rituximab™ are presented in FIG. 12.

The ability of the different derivatives of CytoxB-scFvIg molecules tomediated ADCC was measured using the Bjab B lymphoma cells as the targetand freshly prepared human PBMCs as effector cells. (See Example 2).Effector to target ratios were varied as follows: 70:1, 35:1, and 18:1,with the number of Bjab cells per well remaining constant but the numberof PBMCs were varied. Bjab cells were labeled for 2 hours with ⁵ Cr andaliquoted at a cell density of 5×10⁴ cells/well to each well offlat-bottom 96 well plates. Purified fusion proteins or rituximab wereadded at a concentration of 10 mg/ml to the various dilutions of PBMCs.Spontaneous release was measured without addition of PBMC or fusionprotein, and maximal release was measured by the addition of detergent(1% NP-40) to the appropriate wells. Reactions were incubated for 4hours, and 100 μl of culture supernatant was harvested to a Lumaplate(Packard Instruments) and allowed to dry overnight prior to counting cpmreleased. The results are presented in FIG. 13.

Complement dependent cytotoxicity (CDC) activity of the CytoxBderivatives was also measured. Reactions were performed essentially asdescribed in Example 2. The results are presented in FIG. 14 as percentof dead cells to total cells for each concentration of fusion protein.

Example 6 In Vivo Studies in Macaques

Initial in vivo studies with CytoxB derivatives have been performed innonhuman primates. FIG. 15 shows data characterizing the serum half-lifeof CytoxB in monkeys. Measurements were performed on serum samplesobtained from two different macaques (J99231 and K99334) after doses of6 mg/kg were administered to each monkey on the days indicated byarrows. For each sample, the level of 2H7scFvIg present was estimated bycomparison to a standard curve generated by binding of purifiedCytoxB-(MHWTG1C)-Ig fusion protein to CD20 CHO cells (see Example 2).The data are tabulated in the bottom panel of the FIG. 15.

The effect of CytoxB-(MHWTG1C)Ig fusion protein on levels of circulatingCD40+cells in macaques was investigated. Complete blood counts wereperformed at each of the days indicated in FIG. 16. In addition, FACS(fluorescence activated cell sorter) assays were performed on peripheralblood lymphocytes using a CD40-specific fluorescein conjugated antibodyto detect B cells among the cell population. The percentage of positivecells was then used to calculate the number of B cells in the originalsamples. The data are graphed as thousands of B cells per microliter ofblood measured at the days indicated after injection (FIG. 16).

Example 7 Construction and Expression of an Anti-CD19 scFv-Ig FusionProtein

An anti-CD19 scFv-Ig fusion protein was constructed, transfected intoeukaryotic cells, and expressed according to methods presented inExamples 1, 2, and 5 and standard in the art. The variable heavy chainregions and variable light chain regions were cloned from RNA isolatedfrom hybridoma cells producing antibody HD37, which specifically bindsto CD19. Expression levels of a HD37scFv-IgAHWTG1C and aHD37scFv-IgMHWTG1C were measured and compared to a standard curvegenerated using purified HD37 scFvIg. The results are presented in FIG.17.

Example 8 Construction and Expression of an Anti-L6 scFv-Ig FusionProtein

An scFv-Ig fusion protein was constructed using variable regions derivedfrom an anti-carcinoma mAb, L6. The fusion protein was constructed,transfected into eukaryotic cells, and expressed according to methodspresented in Examples 1, 2, and 5 and standard in the art. Expressionlevels of L6scFv-IgAHWTG1C and L6scFv-IgMHWTG1C were measured andcompared to a standard curve generated using purified HD37 scFvIg. Theresults are presented in FIG. 18.

Example 9 Characterization of Various scFv-IG Fusion Proteins

In addition to the scFv-Ig fusion protein already described, G28-1(anti-CD37) scFv-Ig fusion proteins were prepared essentially asdescribed in Examples 1 and 5. The variable regions of the heavy andlight chains were cloned according to methods known in the art. ADCCactivity of 2H7-MHWTG1C, 2H7-IgAHWTG1C, G28-1-MHWTG1C, G28-1 IgAHWTG1C,HD37-MHWTG1C, and HD37-IgAHWTG1C was determined according to methodsdescribed above (see Example 2). Results are presented in FIG. 19. ADCCactivity of L6scFv-IgAHWTG1C and L6scFv-IgMHWTG1C was measured using the2981 human lung carcinoma cell line. The results are presented in FIG.20. The murine L6 monoclonal antibody is known not to exhibit ADCCactivity.

The purified proteins were analyzed by SDS-PAGE under reducing andnon-reducing conditions. Samples were prepared and gels run essentiallyas described in Examples 2 and 5. The results for the L6 and 2H7 scFv-Igfusion proteins are presented in FIG. 21 and the results for the G28-1and HD37 scFv-Ig fusion proteins are presented in FIG. 22.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for the purposeof illustration, various modifications may be made without deviatingfrom the spirit and scope of the invention. Accordingly, the presentinvention is not limited except as by the appended claims.

1. A method of treatment, comprising administering to a subject in needthereof a composition comprising a pharmaceutically acceptable carrierand a therapeutically effective amount of a single chain protein thatbinds to a disease-associated target, wherein said single chain proteincomprises: (a) a binding domain polypeptide capable of binding to saiddisease-associated target, said binding domain polypeptide being joinedto (b) a hinge peptide, said hinge peptide being joined to (c) animmunoglobulin heavy chain CH2 constant region polypeptide, said CH2constant region polypeptide being joined to (d) an immunoglobulin heavychain CH3 constant region polypeptide, wherein said single chain proteinis capable of mediating at least one immunological activity selectedfrom the group consisting of antibody dependent cell-mediatedcytotoxicity and complement fixation, and wherein said single chainprotein is not radiolabeled.
 2. The method of claim 1 wherein saidsingle chain protein is capable of binding to a cell.
 3. The method ofclaim 2 wherein said cell is a human cell.
 4. The method of claim 1wherein said single chain protein is capable of binding to a receptor.5. The method of claim 4 wherein said receptor is a human receptor. 6.The method of claim 1 wherein said single chain protein is capable ofbinding to a CD antigen.
 7. The method of claim 6 wherein said CDantigen is a human CD antigen.
 8. The method of claim 6 or 7 whereinsaid CD antigen is present on a B cell.
 9. The method of claim 1 whereinsaid binding domain polypeptide is a single chain Fv.
 10. The method ofclaim 9 wherein said single chain Fv is capable of binding to a cell.11. The method of claim 10 wherein said cell is a human cell.
 12. Themethod of claim 9 wherein said single chain Fv is capable of binding toa receptor.
 13. The method of claim 12 wherein said receptor is a humanreceptor.
 14. The method of claim 9 wherein said single chain Fv iscapable of binding to a CD antigen.
 15. The method of claim 14 whereinsaid CD antigen is a human CD antigen.
 16. The method of claim 14 or 15wherein said CD antigen is present on a B cell.
 17. The method of claim1 wherein said hinge peptide is a naturally occurring immunoglobulinhinge peptide.
 18. The method of claim 17 wherein said hinge peptide isan IgG hinge peptide.
 19. The method of claim 18 wherein said hingepeptide is an IgG1 hinge peptide.
 20. The method of claim 17 whereinsaid hinge peptide is an IgA hinge peptide.
 21. The method of claim 20wherein said hinge peptide is an IgA1 hinge peptide.
 22. The method ofany of claims 17-20 or 21, wherein said hinge peptide is human.
 23. Themethod of claim 1 wherein said hinge peptide is an altered IgG hingepeptide having two cysteine residues.
 24. The method of claim 23 whereina hinge peptide cysteine residue has been deleted.
 25. The method ofclaim 23 wherein a hinge peptide cysteine residue has been substitutedwith another amino acid.
 26. The method of claim 25 wherein said aminoacid is serine.
 27. The method of claim 26 wherein said hinge is an SCChinge.
 28. The method of claim 26 wherein said hinge is a CSC hinge. 29.The method of claim 26 wherein said hinge is a CCS hinge.
 30. The methodof claim 1 wherein said hinge peptide is an altered IgG hinge peptidehaving one cysteine residue.
 31. The method of claim 30 wherein one ortwo hinge peptide cysteine residues have been deleted.
 32. The method ofclaim 30 wherein one or two hinge peptide cysteine residues have beensubstituted with other amino acids.
 33. The method of claim 32 whereinat least one of the hinge peptide cysteine resides has been substitutedwith serine.
 34. The method of claim 33 wherein both cysteine resideshave been substituted with serine.
 35. The method of claim 34 whereinsaid hinge is an SSC hinge.
 36. The method of claim 34 wherein saidhinge is an CSS hinge.
 37. The method of claim 34 wherein said hinge isan CSC hinge.
 38. The method of claim 1 wherein said hinge peptide is analtered IgG hinge peptide having no cysteine residues.
 39. The method ofclaim 38 wherein one, two, or three hinge peptide cysteine residues havebeen deleted.
 40. The method of claim 38 wherein one, two, or threehinge peptide cysteine residues have been substituted with other aminoacids.
 41. The method of claim 40 wherein at least one of the hingepeptide cysteine resides has been substituted with serine.
 42. Themethod of claim 41 wherein two hinge peptide cysteine resides have beensubstituted with serine.
 43. The method of claim 41 wherein three hingepeptide cysteine resides have been substituted with serine.
 44. Themethod of any of claims 23-28 or 29 wherein said altered IgG hingepeptide having two cysteine residues is an altered IgG1 hinge peptide45. The method of any of claims 23-28 or 29 wherein said altered IgGhinge peptide having two cysteine residues is an altered human IgG hingepeptide.
 46. The method of claim 45 wherein said altered IgG hinge is anIgG1 hinge.
 47. The method of any of claims 30-36 or 37 wherein saidaltered IgG hinge peptide having one cysteine residue is an altered IgG1hinge peptide.
 48. The method of any of claims 30-36 or 37 wherein saidaltered IgG hinge peptide having one cysteine residue is an alteredhuman IgG hinge peptide.
 49. The method of claim 38 wherein said alteredIgG hinge is an IgG1 hinge.
 50. The method of any of claims 38-42 or 43wherein said altered IgG hinge peptide having no cysteine residues is analtered IgG1 hinge peptide.
 51. The method of any of claims 38-42 or 43wherein said altered IgG hinge peptide having no cysteine residues is analtered human IgG hinge peptide.
 52. The method of claim 51 wherein saidaltered IgG hinge is an IgG1 hinge.
 53. The method of claim 1 whereinsaid hinge peptide is an altered IgA hinge peptide having two cysteineresidues.
 54. The method of claim 1 wherein said hinge peptide is analtered IgA hinge peptide having one cysteine residue.
 55. The method ofclaim 1 wherein said hinge peptide is an altered IgA hinge peptidehaving no cysteine residues.
 56. The method of any of claims 53, 54, or55, wherein at least one hinge peptide cysteine residue has beendeleted.
 57. The method of any of claims 53, 54, or 55, wherein anotheramino acid has been substituted for at least one hinge peptide cysteineresidue.
 58. The method of claim 57 wherein said amino acid is serine.59. The method of any of claims 53, 54, or 55, wherein said altered IgAhinge is an altered IgA1 hinge.
 60. The method of claim 59 wherein saidaltered IgA1 hinge is an altered human IgA1 hinge.
 61. The method ofclaim 1 wherein said heavy chain CH2 constant region polypeptide is anIgG CH2 constant region polypeptide.
 62. The method of claim 1 whereinsaid heavy chain CH2 constant region polypeptide is an IgG1 CH2 constantregion polypeptide.
 63. The method of claim 1 wherein said heavy chainCH2 constant region polypeptide is an IgA CH2 constant regionpolypeptide.
 64. The method of claim 1 wherein said heavy chain CH2constant region polypeptide is an IgA1 CH2 constant region polypeptide.65. The method of any of claims 61-63, or 64, wherein said CH2 constantregion polypeptide is human.
 66. The method of claim 1 wherein saidheavy chain CH2 constant region polypeptide is an altered CH2 constantregion derived from a human heavy chain CH2 constant region polypeptide.67. The method of claim 66, wherein said heavy chain constant regioncomprises a CH2 domain in which the amino acid at position 234 has beendeleted or replaced with another amino acid.
 68. The method of claim 67,wherein leucine has been replaced with serine at position
 234. 69. Themethod of claim 1 wherein said heavy chain CH3 constant regionpolypeptide is an IgG CH3 constant region polypeptide.
 70. The method ofclaim 1 wherein said heavy chain CH3 constant region polypeptide is anIgG1 CH3 constant region polypeptide.
 71. The method of claim 1 whereinsaid heavy chain CH3 constant region polypeptide is an IgA CH3 constantregion polypeptide.
 72. The method of claim 1 wherein said heavy chainCH3 constant region polypeptide is an IgA1 CH3 constant regionpolypeptide.
 73. The method of any of claims 69-71 or 72 wherein saidCH3 constant region polypeptide is human.
 74. The method of claim 1wherein said heavy chain CH2 and CH3 constant region polypeptides areIgG CH2CH3 constant region polypeptides.
 75. The method of claim 1wherein said heavy chain CH2 and CH3 constant region polypeptides areIgG1 CH2CH3 constant region polypeptides.
 76. The method of claim 1wherein said heavy chain CH2 and CH3 constant region polypeptides areIgA CH2CH3 constant region polypeptides.
 77. The method of claim 1wherein said heavy chain CH2 and CH3 constant region polypeptides areIgA1 CH2CH3 constant region polypeptides.
 78. The method of any ofclaims 74-76 or 77 wherein said CH2 and CH3 constant region polypeptidesare human.
 79. The method of claim 1 wherein said heavy chain CH3constant region polypeptide is an altered CH3 constant region derivedfrom a human heavy chain CH3 constant region polypeptide.
 80. The methodof claim 1 wherein said single chain protein is chimeric.
 81. The methodof claim 80 wherein said subject is human.
 82. The method of any ofclaims 2-7, 9-14 or 15, wherein said single chain protein is chimeric.83. The method of claim 82 wherein said subject is human.
 84. The methodof claim 1 wherein said single chain protein is humanized.
 85. Themethod of claim 84 wherein said subject is human.
 86. The method of anyof claims 2-7, 9-14 or 15, wherein said single chain protein ishumanized.
 87. The method of claim 86 wherein said subject is human. 88.The method of claim 1 wherein said single chain protein is designed tominimize immunogenicity upon administration to said subject.
 89. Themethod of claim 88 wherein said subject is human.
 90. The method of anyof claims 2-7, 9-14 or 15, wherein said single chain protein is designedto minimize immunogenicity upon administration to said subject.
 91. Themethod of claim 90 wherein said subject is human.